Login| Sign Up| Help| Contact|

Patent Searching and Data


Title:
METHOD FOR QUANTIFICATION OF MULTIPLE PROTEINS IN A SAMPLE
Document Type and Number:
WIPO Patent Application WO/2021/006747
Kind Code:
A1
Abstract:
The invention relates to an analytical method for identification and quantification of multiple proteins in a sample. Although the present invention can be applied to many proteins, particular reference will be made to bioactive proteins. In particular, although not exclusively, the invention relates to an efficient method for identifying and quantifying two or more bioactive proteins in a sample. The sample is typically derived from milk, and may be a particular fraction derived from milk or a product containing the foregoing.

Inventors:
CLAYCOMB RODNEY WAYNE (NZ)
OGLE COLIN ROGER (NZ)
ADAM KATHARINE HELEN (NZ)
SMOLENSKI GRANT ALAN (NZ)
Application Number:
PCT/NZ2020/050063
Publication Date:
January 14, 2021
Filing Date:
July 03, 2020
Export Citation:
Click for automatic bibliography generation   Help
Assignee:
QUANTEC LTD (NZ)
International Classes:
G01N33/68; G01N33/04
Domestic Patent References:
WO2017183996A12017-10-26
Other References:
VINCENT DELPHINE, EZERNIEKS VILNIS, ELKINS AARON, NGUYEN NGA, MOATE PETER J., COCKS BENJAMIN G., ROCHFORT SIMONE: "Milk bottom-up proteomics: method optimization", FRONTIERS IN GENETICS, vol. 6, no. 360, 11 January 2016 (2016-01-11), pages 1 - 24, XP055785676, DOI: 10.3389/fgene.2015.00360
BAR, C. ET AL.: "Protein profile of dairy products: Simultaneous quantification of twenty bovine milk proteins", INTERNATIONAL DAIRY JOURNAL, vol. 97, 18 January 2019 (2019-01-18), pages 167 - 175, XP085796921, Retrieved from the Internet DOI: 10.1016/j.idairyj.2019.01.001
MURATA M., WAKABAYASHI H., YAMAUCHI K., ABE F.: "Identification of milk proteins enhancing the antimicrobial activity of lactoferrin and lactoferricin", JOURNAL OF DAIRY SCIENCE, vol. 96, no. 8, August 2013 (2013-08-01), pages 4891 - 4898, XP055785678
BRICK TABEA, EGE MARKUS, BOEREN SJEF, BÖCK ANDREAS, VON MUTIUS ERIKA, VERVOORT JACQUES, HETTINGA KASPER: "Effect of processing intensity on immunologically active bovine milk serum proteins", NUTRIENTS, vol. 9, no. 9, 31 August 2017 (2017-08-31), pages 963 - 14, XP055785680, DOI: 10.3390/nu9090963
ZHANG, J. ET AL.: "Determination of bovine lactoferrin in dairy products by ultra-high performance liquid chromatography-tandem mass spectrometry based on tryptic signature peptides employing an isotope-labeled winged peptide as internal standard", ANALYTICA CHIMICA ACTA, vol. 829, 2014, pages 33 - 39, XP029026947, Retrieved from the Internet DOI: 10.1016/j.aca.2014.04.025
Attorney, Agent or Firm:
SCOTT, Andrew et al. (NZ)
Download PDF:
Claims:
Claims

1. Use of mass spectrometry to identify and quantify fragments of two or more proteins in a milk protein sample.

2. The use according to claim 1, wherein two or more proteins are selected from

lactoferrin (LTF), lactoperoxidase (LPO), Lysosomal alpha mannosidase (LAM),

Immunoglobulin G (IgG), Ribonuclease (Rnase), and/or Angiogenin (Ang).

3. A method for quantifying two or more proteins in a sample that contains milk protein(s), the method comprising:

providing a sample containing a milk protein;

treating the sample with a digestive enzyme to form digested peptide fragments; and

analysing the digested peptide fragments by mass spectrometry to quantify two or more proteins present in the sample.

4. A method according to claim 3, wherein the two or more proteins are selected from lactoferrin (LTF), lactoperoxidase (LPO), Lysosomal alpha mannosidase (LAM),

Immunoglobulin G (IgG), Ribonuclease (Rnase), and/or Angiogenin (Ang).

5. A method according to claim 3 or 4, wherein three or more proteins are quantified by mass spectrometry.

6. A method according to claim 3 or 4, wherein four or more proteins are quantified by mass spectrometry.

7. A method according to claim 3 or 4, wherein five or more proteins are quantified by mass spectrometry.

8. A method according to claim 3 or 4, wherein six or more proteins are quantified by mass spectrometry.

9. A method according to any one of claims 3 to 8, wherein analysis of the digested fragment is by tandem mass spectrometry.

10. A method according to any one of claims 3 to 9, wherein at least one protein quantified is selected from lactoferrin or lactoperoxidase.

11. A method according to any one of claims 3 to 10, wherein the proteins quantified include lactoferrin and lactoperoxidase.

12. A method as claimed in any one of claims 3 to 11, wherein the peptide fragment(s) analysed are selected from:

13. A method as claimed in claim 12, wherein the method further comprises analysing a daughter ion of the peptide fragment selected from:

14. A method for identifying and determining bioactive properties of a sample that contains milk protein(s), the method comprising: providing a sample containing milk protein(s);

preparing the sample for analysis; and

analysing the sample by quantification of two or more bioactive proteins using mass spectrometry.

15. A method according to claim 14, wherein the quantification of two or more proteins are selected from lactoferrin (LTF), lactoperoxidase (LPO), Lysosomal alpha mannosidase (LAM), Immunoglobulin G (IgG), Ribonuclease (Rnase), and/or Angiogenin (Ang).

16. A method according to claim 14 or 15, wherein three or more proteins are quantified by mass spectrometry.

17. A method according to claim 14 or 15, wherein four or more proteins are quantified by mass spectrometry.

18. A method according to claim 14 or 15, wherein five or more proteins are quantified by mass spectrometry.

19. A method according to claim 14 or 15, wherein six or more proteins are quantified by mass spectrometry.

20. A method according to any one of claims 14 to 19, wherein quantification of the two or more proteins is by tandem mass spectrometry.

21. A method according to any one of claims 14 to 20, wherein at least one of the proteins quantified are selected from lactoferrin or lactoperoxidase.

22. A method according to any one of claims 14 to 21, wherein the proteins quantified include lactoferrin and lactoperoxidase.

23. A method for identifying, determining and indexing bioactive properties of a sample containing a milk protein, the method comprising:

i. providing and preparing a sample containing a milk protein for analysis;

ii. analysing the sample to identify two or more bioactive proteins using mass spectrometry;

iii. optionally, subjecting the sample to a further activity and/or a specific binding test to establish the magnitude of a bioactive property of one or more milk protein(s) in the sample; and

iv. indexing the sample containing the milk protein based on results obtained from steps ii and iii.

24. A method according to claim 23, wherein the bioactive proteins to be identified are selected from lactoferrin (LTF), lactoperoxidase (LPO), Lysosomal alpha mannosidase (LAM), Immunoglobulin G (IgG), Ribonuclease (Rnase), and /or Angiogenin (Ang).

25. A method according to claim 23 or 24, wherein three or more proteins are identified.

26. A method according to claim 23 or 24, wherein four or more proteins are identified.

27. A method according to claim 23 or 24, wherein five or more proteins are identified.

28. A method according to claim 23 or 24, wherein six or more proteins are identified.

29. A method according to any one of claims 23 to 28, wherein at least one of the proteins identified include are lactoferrin or lactoperoxidase.

30. A method according to any one of claims 23 to 29, wherein the proteins identified include lactoferrin and lactoperoxidase.

31. A method according to any one of claims 23 to 31, wherein preparation of the sample for analysis includes treating the sample with a digestive enzyme to form a digested peptide fragment.

32. A method according to claim 31, wherein the identification bioactive proteins include quantification of the peptide fragments using mass spectrometry.

33. A method according to any one of claims 32, wherein the quantification of the peptide fragments uses tandem mass spectrometry.

34. A method as claimed in claim 33, wherein the peptide fragment(s) quantified are selected from:

35. A method as claimed in claim 34, wherein the method further comprises analysing a daughter ion of the peptide fragment selected from:

36. A method according to any one of claims 23 to 35, wherein the enzyme activity test is a lactoperoxidase activity test.

37. A method according to any one of claims 23 to 36, wherein the antimicrobial activity test is screened against Escherichia coli and/or Staphylococcus aureus.

38. Use of one or more signature peptide fragment(s) to identify and quantify two or more proteins using mass spectrometry.

39. Use according to claim 38, wherein the signature peptide fragment(s) are selected from the group consisting of: LRPVAAEIYGTK (SEQ ID No 7), SVDGKEDLIWK (SEQ ID No 8); ILGAFIQIITFR (SEQ ID No 9), DGGIDPLVR (SEQ ID No 10), GLQTVGLK (SEQ ID No 11); WGPETLLLR (SEQ ID No 12), FQVIVYNPLGR (SEQ ID No 13); EPQVYVLAPPQEELSK (SEQ ID No 14), VHN EGLPAPIVR (SEQ ID No 15), VVSALR (SEQ ID No 16), YIHFLTQHYDAKPK (SEQ ID No 17), NTFIHGNK (SEQ ID No 18) or FNTFIHEDLWN IR (SEQ ID No 19).

Description:
METHOD FOR QUANTIFICATION OF MULTIPLE PROTEINS IN A SAMPLE

Field of Invention

The invention relates to an analytical method for identification and quantification of multiple proteins in a sample.

Although the present invention can be applied to many proteins, particular reference will be made to bioactive proteins.

In particular, although not exclusively, the invention relates to an efficient method for identifying and quantifying two or more bioactive proteins in a sample. The sample is typically derived from milk, and may be a particular fraction derived from milk or a product containing the foregoing.

Background to the Invention

Milk is a nutrient-rich product containing proteins, lipids, minerals and lactose.

The popularity and availability of processed dairy-based products derived from milk such as milk powder, milk protein powder or whey powder for human consumption is increasing globally.

The dairy-based products provide a convenient source of nutrition. Recent studies have identified a group of proteins in these products as having beneficial bioactive properties, such as lactoferrin, lactoperoxidase and angiogenin (Ribonuclease), which may have activity against bacteria, yeast, fungi and/or viruses. However, the presence of these proteins varies from product to product due to a variety of factors such as source of the milk, processing environment, formulation or degradation of product. The dairy-based supplements and health food sector is lacking an effective method to identify and quantify the bioactive properties of finished commercial products, which would provide consumers and manufacturers an effective tool to distinguish the numerous products already on the market.

In the applicant's earlier application - WO2017183996, it was recently found that certain combinations of proteins found in milk derived products, particularly in the cationic fraction, were found to provide therapeutic properties. In particular, the combinations were found to have surprising selectivity towards inhibition of pathogenic micro-organisms compared to a considerably less inhibitory effect towards beneficial commensal micro-organisms that are present in a healthy microbiome.

However, identifying and quantifying two or more beneficial bioactive proteins in a sample has proven to be a difficult and time-consuming task.

Traditionally, methods for identifying target proteins in a sample have been limited to identifying a single specific protein at a time. In order to analyse multiple proteins in a sample, multiple tests on the same sample would need to be undertaken. Such a process is not conveniently scalable to testing the vast number of milk derived products that are obtained daily from milk which can be starkly different depending on the source of the milk, processing environment, formulation or degradation of product.

It is an object of the invention to address the foregoing problems or at least provide the public with a useful choice.

Summary of the Invention

According to one aspect of the invention, there is provided a use of mass spectrometry to identify and quantify fragments of two or more proteins in a milk derived protein sample. The present inventors have determined that mass spectrometry provides an efficient method of identifying and quantifying peptide fragments of two or more proteins in a milk protein sample. The proteins of particular interest include lactoferrin (LTF), lactoperoxidase (LPO), Lysosomal alpha mannosidase (LAM), Immunoglobulin G (IgG), Ribonuclease (Rnase), and/or Angiogenin (Ang); some or all of which may be present in a sample of milk proteins. It should also be noted that sub-classes of these proteins may also exist, such as Rnase-1, Rnase-4, IgG-l, lg-2, etc.

To assist in the identification and quantification of fragments of two or more proteins in a milk derived sample, the inventors have identified unique non-overlapping peptide sequences for each of the target proteins of interest which helps enable the efficient identification and quantification of proteins in a milk derived sample. The identified sequences were then used to identify specific unique peptide fragments of each protein of interest to then devise the method of the present invention.

While it is known that a combination of two or more of these proteins can provide a therapeutic effect, it is time consuming and difficult to determine whether a particular source of milk protein will contain two or more these proteins and/or how much of these proteins are included in the milk protein source.

The inventors have spent considerable time to identify specific, unique peptide fragments within the specified proteins of interest. In particular, it was important that there were no overlapping sequences present that could cause confusion as to the amount or presence of the individual proteins and/or lead to false positives or incorrect results.

The inventors have also devised methods by which those specific, unique peptide fragments can be extracted from a sample using a specialised digestive process they have developed.

The end result of the inventors' efforts around the extraction of the specific fragments has led to use of these fragments as indicators of proteins of interest which may be easily and quickly identified and quantified within a sample of milk derived protein or a sample that contains a milk-derived protein.

These methods, utilising mass spectrometry, allow for a large number of different milk derived protein samples or samples containing milk-derived protein to be analysed in an efficient and timely manner, in order to identify and quantify the peptide fragments of interest and thereby identify and quantify the proteins themselves.

In turn, this invention addresses the problem of first determining whether a given sample that has milk proteins of a particular type, and secondly determining the likely magnitude of the bioactivity of the selected proteins within that sample.

According to one aspect of the invention, there is provided a method for quantifying two or more proteins in a sample that contains milk protein(s), the method comprising the steps of: providing a sample that contains milk protein(s);

treating the sample with a digestive enzyme to form digested peptide fragments; and analysing the digested peptide fragments by mass spectrometry to quantify two or more proteins present in the sample,

the two or more proteins being selected from:

i) lactoferrin (LTF);

ii) lactoperoxidase (LPO);

iii) Lysosomal alpha mannosidase (LAM);

iv) Immunoglobulin G (IgG);

v) Ribonuclease (Rnase); and/or

vi) Angiogenin (Ang).

While the methods of the invention may be used to quantify two or more proteins in a sample that contains milk protein(s), it will be understood that the efficiency of the method is particularly well suited to the identification of three of more proteins in a sample, such as four or more proteins in a sample, for instance five or more proteins in a sample, or alternatively such as six or more proteins in a sample. It is believed that the methods of the invention allow the quantification of a multitude of proteins in a milk protein sample for the first time, particularly in such an efficient manner.

In particular it is believed that the presence of lactoferrin and/or lactoperoxidase are especially beneficial and provide unique bioactive properties to a product such as a milk derived product.

According to one aspect of the invention, there is provided a method for quantifying lactoferrin (LTF) and one or more additional milk protein(s) in a sample that contains a milk protein(s), the method comprising the steps of:

providing a sample containing milk protein(s);

treating the sample with a digestive enzyme to form digested peptide fragments; and analysing the digested peptide fragments by mass spectrometry to quantify lactoferrin and one or more additional protein(s) present in the sample,

wherein the additional protein(s) being selected from lactoperoxidase (LPO), Lysosomal alpha mannosidase (LAM), Immunoglobulin G (IgG), Ribonuclease (Rnase), and/or Angiogenin (Ang).

According to one aspect of the invention, there is provided a method for quantifying lactoperoxidase (LPO) and one or more additional milk protein(s) in a sample that contains a milk protein(s), the method comprising the steps of:

providing a sample containing milk protein(s);

treating the sample with a digestive enzyme to form a digested peptide fragment, and analysing the digested peptide fragment by mass spectrometry to quantify

lactoperoxidase and one or more additional protein(s) present in the sample, wherein the additional protein(s) being selected from lactoferrin (LTF), Lysosomal alpha mannosidase (LAM), Immunoglobulin G (IgG), Ribonuclease (Rnase), and/or Angiogenin (Ang).

It will be appreciated that while peptide fragments can be used to identify and quantify the specific target proteins using mass spectrometry, more broadly, it would be understood that quantification of proteins can also be achieved using mass spectrometry also. According to a further aspect of the invention, there is provided a method for determining bioactive properties of a milk derived protein sample, the method comprising the steps of: providing a milk derived protein sample;

preparing the protein sample for analysis;

analysing the sample by quantification of two or more bioactive proteins using mass spectrometry;

wherein the bioactive proteins are selected from lactoferrin (LTF), lactoperoxidase (LPO), Lysosomal alpha mannosidase (LAM), Immunoglobulin G (IgG), Ribonuclease (Rnase), and/or Angiogenin (Ang).

It has been determined by the inventors that the identification and quantification of two or more bioactive proteins in a product, such as a milk derived product or a milk protein product, or generally the quantification of three or more proteins in a product, or the quantification of four or more proteins in a product, or the quantification of five or more proteins in a product, or particularly the quantification of six or more proteins in a product provides a useful measure for identifying beneficial bioactive properties of a product. Individually the bioactive proteins are known to have therapeutic properties and it is believed that the combination of two or more of these proteins of interest can provide a therapeutic effect.

In a further aspect of the invention, the methods of the invention may include the additional step of subjecting the milk derived protein sample to at least one additional test to establish a further bioactive property of the sample. Such tests may include, but are not limited to, enzyme activity test and/or an antimicrobial test. Without wishing to be bound by theory, it is believed that the use of the at least one additional test is helpful in providing a further level of confidence in the bioactive properties of the milk derived product. Such tests provide a useful indicator to assess whether the milk protein(s) have retained their bioactive properties during the manufacturing and/or formulation process of the milk protein-based product. Measuring the specific bioactivity of one or more proteins may allow a level of confidence of the presence of one or more milk-derived protein(s) retaining its bioactive properties in the milk derived product.

The use of the at least one additional test may also be helpful in calibrating the quantification data provided by the step of analysing the digested peptide fragments by mass spectrometry to quantify two or more proteins present in the sample, so as to provide a further level of confidence in the bioactive properties of the milk derived protein product.

According to a further aspect of the invention, there is provided a method for determining bioactive properties of a milk protein product, the method comprising the steps of:

providing a milk protein sample;

preparing the sample for analysis;

analysing the sample to quantify of two or more proteins using mass spectrometry; subjecting the sample to an additional activity test to establish the magnitude of a bioactive property of one or more protein(s) in the sample;

wherein the bioactive proteins to be quantified are selected from lactoferrin (LTF), lactoperoxidase (LPO), Lysosomal alpha mannosidase (LAM), Immunoglobulin G (IgG),

Ribonuclease (Rnase), and/or Angiogenin (Ang).

According to a further aspect of the invention, there is provided a method for determining and indexing bioactive properties of a sample that contains milk protein, the method comprising: i. providing and preparing a sample containing a milk protein for analysis;

ii. analysing the sample to quantify of two or more proteins using mass spectrometry; iii. optionally, subjecting the product to a further activity test to establish the magnitude of a bioactive property of one or more milk protein(s) in the sample; and

iv. indexing the sample containing the milk protein(s) based on results obtained from steps ii and iii;

wherein the bioactive proteins to be quantified are selected from lactoferrin (LTF),

lactoperoxidase (LPO), Lysosomal alpha mannosidase (LAM), Immunoglobulin G (IgG),

Ribonuclease (Rnase), and/or Angiogenin (Ang). It will be understood that the technique of mass spectrometry is a powerful tool that can be used to fragment and analyse components within a mixture. The methods of the present invention are particularly well suited to tandem mass spectrometry techniques (MS/MS). In certain embodiments of the methods disclosed herein, mass spectrometry is performed in positive ion mode. Alternatively, mass spectrometry may be performed in negative ion mode.

Various ionization sources, including for example electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI), may be used in embodiments of the present invention.

In certain embodiments the methods of the present invention involve the use of mass spectrometry in sequence with a chromatographic technique, such as liquid chromatography.

In preferred embodiments, the liquid chromatography is high performance liquid

chromatograph (HPLC). In more preferred embodiments, the liquid chromatography is reverse- phase high performance liquid chromatograph (RP-HPLC).

Further aspects of the invention, which should be considered in all its novel aspects, will become apparent to those skilled in the art upon reading of the following description which provides at least one example of a practical application of the invention.

Brief Description of the Drawings

One or more embodiments of the invention will be described below by way of example only, and without intending to be limiting, with reference to the following drawings, in which:

Figure 1: Shows a GS900 scan of Coomassie stained SDS-PAGE gel with annotated

molecular weight bands and sample lanes. Lane 1; SeeBlue plus2 molecular weight standard (Mwt), Lane 3; IDP Milk protein powder (MPP 60 pg), Lane 5; Strawberry flavoured tablet (Straw; 60 pg), Lane 7; Mint flavoured lozenge (Mint; 30 pg). Figure 2: Selection of protein bands for in-gel tryptic digestion. (A) Sections of individual lanes covering the proteins of interest were excised based on the position of the SeeBlue 2 molecular weight marker (Mwt, lane 1). (B) Each gel slice was further divide into multiple slivers, as indicated, for efficient removal of Coomassie stain, reduction, alkylation, and tryptic digestion.

Figure 3: Shows targeted LAM peptides in milk protein powder from Slice-1, identified using the Thermo Xcalibur Qual Browser.

Figure 4: Shows peptides identified by LC-MS/MS mapped to the bovine lactoferrin

sequence.

Figure 5: Shows peptides identified by LC-MS/MS mapped to the bovine lactoperoxidase sequence.

Figure 6: Shows peptides identified by LC-MS/MS mapped to the bovine lysosomal alpha- mannosidase sequence.

Figure 7: Shows peptides identified by LC-MS/MS mapped to the bovine Immunoglobulin gammal heavy chain sequence.

Figure 8: Shows peptides identified by LC-MS/MS mapped to the bovine angiogenin-1 sequence.

Figure 9: Shows peptides identified by LC-MS/MS mapped to the bovine RNase4

sequence.

Figure 10: Shows the gradient conditions for the LC-MS/MS. Figure 11: Shows typical linear calibration curves with a 1/x weighting for quantifying (QT) ions for each protein of interest.

Figure 12: Shows the typical total ion count (TIC) chromatogram of base powder 0/0 pg/L calibration standard containing 200 ppm ISTD.

Figure 13: Shows growth curves for K. aerogenes and S. aureus in wells of a 96 well plate with measurement based on turbidity (ODsoo) with reads taken every 20 minutes for 18 hours. K. aerogenes wells A1 to Dl: media only +ve controls, A2 to D2 media + NaSCN +ve controls, El to HI media only -ve controls, E2 to H2 media + NaSCN -ve controls, A3 to H3 product dilutions -ve controls, A4-5-6 to H4-5-6 sample dilutions. S. aureus wells A12 to D12 media only +ve controls, All to Dll media + NaSCN +ve controls, E12 to H12 media only -ve controls, Ell to Hll media + NaSCN -ve controls, A10 to H10 product dilutions -ve controls, A7-8-9 to H7-8-9 sample dilutions.

Figure 14: Shows growth curves for S. aureus growth in wells of a 96 well plate with

measurement based on turbidity (Oϋ q oo) with reads taken every 20 minutes for 18 hours. Wells A1 to Dl: media only +ve controls, A2 to D2 media + NaSCN +ve controls, El to H I media only -ve controls, E2 to H2 media + NaSCN -ve controls, A3 to H3 product dilutions -ve controls, A4-5-6 to H4-5-6 sample 1 dilutions, A7- 8-9 to H7-8-9 sample 2 dilutions, AlO-11-12 to H lO-11-12 sample 3 dilutions. The highest dilution of sample is in the wells in row A and the lowest in row H.

Figure 15: Shows growth curves for K. aerogenes and S. aureus in wells of a 96 well plate with measurements based on turbidity (OD600) with reads taken every 20 minutes for 18 hours. K. aerogenes wells A1 to Dl: media only +ve controls, A2 to D2 media + NaSCN -i-ve controls, El to HI media only -ve controls, E2 to H2 media + NaSCN -ve controls, A3 to H3 product dilutions -ve controls, A4-5-6 to H4-5-6 sample dilutions. S. aureus wells A12 to D12 media only +ve controls, All to Dll media + NaSCN +ve controls, E12 to H12 media only -ve controls, Ell to Hll media + NaSCN -ve controls, A10 to H 10 product dilutions -ve controls, A7- 8-9 to H7-8-9 sample dilutions. The sample used was lactoferrin.

Detailed description of the invention

It will be appreciated the term "sample" or "test sample" as used herein is any substance that contains a component derived or separated from an animal-based product such as milk, processed milk powders, milk protein powders, cationic whey protein powders or the like.

As used herein the term "milk derived" includes any component or sample that is derived, extracted or isolated from whole milk, skim milk, fat-free milk, low fat milk, full fat milk, lactose-free or lactose-reduced milk (produced by hydrolysing the lactose by lactase enzyme to glucose and galactose, or by other methods such as nanofiltration, electrodialysis, ion exchange chromatography and centrifugation technology), concentrated milk or dry milk.

It will be appreciated the term "product" as used herein is any finished component in which a sample may be taken for analysis.

WO/2017/183996, the entire contents of which is incorporated herein by reference, discloses useful combinations of proteins extracted and isolated from a cationic fraction of milk. It has been found that some or all the proteins in the cationic fraction isolated from milk may collectively work together to provide highly beneficial bioactive activity to a finished product.

Throughout this specification, use of the term 'cationic whey proteins' or 'cationic fraction' should be taken as meaning a fraction or isolated components from a milk, being components that have an isoelectric point of or above substantially 6.8. Techniques for fractionating or isolating the components from milk may include those disclosed in WO/2017/183996 and include isolating the cationic components following binding to cation exchange media.

However, it will be appreciated that other methods or techniques for fractioning and/or isolating components, such as proteins from milk can be used with used with the method of the present invention.

The cationic whey proteins as used herein, may be isolated or extracted from one or more sources of milk, such as bovine milk, sheep milk, goat milk, buffalo milk, camel milk, human milk and the like. The major and minor proteins found in bovine milk (used for this preliminary study) are also found in other sources of milk, with very similar isoelectric points in each case. Additionally, the term milk should be taken to include colostrum, whole milk, skim milk or whey.

It should be appreciated that one of the reasons for deriving the present invention came from the desire to determine quickly the likely bio-activity of products containing the inventors' cationic fraction as specified in WO/2017/183996. With the introduction of products on the market from other sources claiming similar bio-activity, it became apparent that the present invention could also be used to test those products as well as the veracity of the product claims. While some products do contain one or more milk proteins of interest, the presence of such protein(s) does not guarantee that the milk protein(s) are still active nor active to a specific extent. For example, spray-drying of milk protein fractions can denature the milk proteins rendering them inactive. Although assays for the presence of the proteins would still result in a positive presence, the denaturing process has rendered such proteins inactive. Thus, the present invention can be used to identify premiere products and fraudulent claims and also to allow consumers to know if the product being purchased still retains its activity. The present invention can also be used to test shelf-life of products in order to determine if the bioactivity of the milk proteins present at the time of manufacture has remained over time.

The most prevalent proteins in the samples of interest according to the present invention include lactoferrin (LTF), lactoperoxidase (LPO), Lysosomal alpha mannosidase (LAM),

Immunoglobulin G (IgG), Ribonuclease (Rnase) and/or Angiogenin (Ang). There are also a wide number of additional proteins in milk which may also have bioactive properties, and which may be isolated as part of the cationic fractions, and as such it should not be seen to be limited to the proteins identified above.

Without limitation, proteins of interest that can be found in the cationic fraction of milk are discussed in more detail below. It should be appreciated that many of these proteins are believed to be associated with an innate immune response and/or impart some level of bioactive property in a finished product formulation.

Lactoperoxidase

Lactoperoxidase (Lp) is a protein present in the mammary gland secretion and many other exocrine secretions of mammals.

The Lactoperoxidase system consists of three components— Lp, thiocyanate and hydrogen peroxide, which are all present in fresh milk. Lp catalyses the oxidation of thiocyanate by peroxide and generates intermediate products (hypothiocyanite (OSCN-)), with antibacterial properties. Thiocyanate is present in the mammary, salivary and thyroid glands and their secretions, in synovial, cerebral, cervical and spinal fluids, in lymph and plasma, and in organs such as stomach and kidney. Hydrogen peroxide, the third component of the Lactoperoxidase system is not normally detected in milk, but is present during infection.

The Lactoperoxidase system has bacteriostatic or bactericidal activity on a variety of susceptible micro-organisms including bacteria, fungi and viruses associated with mastitis.

Lactoferrin

Lactoferrin (Lf) is a glycoprotein which is present in mammary gland secretion and many other exocrine secretions of mammals. Lf is secreted predominately by surface epithelia into the mucosal environment. Lactoferrin is a multifunctional protein that has antibacterial, antifungal, antiviral, antitumour, anti-inflammatory, and immunoregulatory properties. Therefore, the inventors believe that Lactoferrin contributes bioactive properties, such as anti microbial effects to a product, but more importantly in combination with at least one other protein, imparts an additional level of selectivity towards pathogenic micro-organisms yet with very low MIC levels towards commensals.

Lf is produced at high levels in nasal and tracheal passages, and in gastric, genital and ophthalmic secretions. Lf is also produced at high levels in neutrophils where it is stored in secondary granules and released during inflammation.

The mechanism by which Lf inhibits microbial growth is yet to be fully established. Its antimicrobial and anti-inflammatory effects are believed to be as a result of a number of different actions or functions of Lf.

The highly basic N terminal region of bovine lactoferrin is thought to be essential for antimicrobial activity. The 25 N-terminal amino acids may be removed by proteases to form lactoferricin (Lfcin). These proteases may be naturally occurring in milk or serum, and many micro-organisms produce proteases. Lfcin is up to a 1000-fold more effective against some micro-organisms than intact lactoferrin. Lfcin has been shown to inhibit a diverse range of microorganisms such as gram-negative bacteria, gram-positive bacteria, yeast, filamentous fungi, and parasitic protozoa, including some antibiotic-resistant pathogens. Therefore, it is plausible that lactoferricin may be added to the combination, such as the composition, replace lactoferrin, and/or be a natural degradation product of lactoferrin in the combination of the present invention due to proteolytic action.

Angiogenin-Ribonuclease

Angiogenin-Ribonuclease belongs to the ribonuclease superfamily have been identified in milk and is known to have some anti-viral and anti-microbial activity. Therefore, it is envisaged that Angiogenin-Ribonuclease contributes bioactive properties, such as anti-microbial effects, to a product, such as cationic whey proteins, but more importantly is somehow (in combination with the other protein(s) in combination) helping to impart an intricate level of selectivity towards pathogenic micro-organisms yet with very low M IC levels towards commensals.

Lysozyme-like proteins, such as chitinase-like protein (CLP-1) or lysosomal alpha mannosidase (LAM)

Lysozyme-like protein, such as chitinase-like protein (CLP-1) or lysosomal alpha mannosidase (LAM) may also be detected in a product by the method described herein. Lysozyme-like proteins (such as CLP-1 or LAM) have cell lysing activity and thereby are thought to enhance antimicrobial activity through their lysozyme-like effects.

Immunoglobulins

Immunoglobulins are important components of milk and provide passive protection to the suckling young. Although they are not strongly cationic some immunoglobulins, IgG, IgM, IgA and polymeric immunoglobulin receptor (PIGR) are captured by cation exchange.

Immunoglobulins are important in the first line of defence against foreign invaders.

Immunoglobulins bind to micro-organisms and thus opsonise them so that they are more easily recognized by phagocytic cells. It is plausible, therefore, that they may also have some effect on the observed selectivity in the present invention and may be working synergistically with other proteins in the cationic fraction.

It is believed that some or all the proteins of interest identified in the cationic fraction isolated from milk collectively work together to somehow induce highly beneficial selectively towards numerous pathogenic micro-organisms without a comparative level of inhibition of commensals. Further testing is being conducted to determine which particular combination(s) provide the best results; however initial trials have indicated that selectively is synergistically enhanced if retaining more proteins from the cationic fraction of milk together.

It is envisaged that other milk proteins may also be quantified by the method described herein. Proteins that include bioactive properties which can improve effectiveness of a product (either through imparting selectivity, or some other form of indirectly modulation of the protein(s) functionality) being particularly preferred include:

- cathelicidin 1;

- N-acetyl glucosaminidase;

- serum amyloid A;

- b Defensin;

- Peptidoglycan recognition protein;

- Xanthine dehydrogenase;

- Immunoglobulin(s) IgA, IgD, IgM, IgA, and/or IgE;

- Growth factors EGF, IGF 1, TGF B1 and TGF B2.

It will be appreciated that some of the cationic fraction components (e.g. lactoferrin, angiogenin) may also have minor variants,—such as variations in amino acid sequence or in degree and type of glycosylation, these minor variants, and their presence in the cationic fraction should also be taken as being covered by the present application.

The term "digestive enzyme" as used herein is an enzyme capable of cleaving or hydrolysing peptides or proteins into fragments in a specific or generic, random manner, for example. Such enzymes may be referred to as proteolytic enzymes, otherwise known as peptidases, proteases and proteinases. Such enzymes may include aspartic proteases, cysteine proteases, glutamic proteases, metalloproteases, asparagine proteases, serine proteases, threonine proteases, and proteases with mixed or unknown catalytic mechanism. A digestive enzyme can form a digested peptide fragment from a protein, including where the protein is a component of a sample. Digestive enzymes include proteases such as trypsin, chymotrypsin, enterokinase,

endoproteinase, elastase, subtilisin, proteinase K, thrombin factor Xa, WNV protease, bromelain, papain, ficin (ficain), rhinovirus 3C, TEV protease, TVMV protease, thermolysin, collagenase, dispase, pepsin, cathepsin D, carboxypeptidases, cathepsin C, and DAPase. A preferred digestive enzyme is trypsin. The term "preparation" or "preparing" as used herein, refers to any procedure, method or work up required or performed on a sample or product to make it suitable for analysis.

The term "protein" as used herein, refers to a polymer of amino acids, not having any specific length. Thus, peptides (including oligopeptides and polypeptides) and protein fragments are included within this definition.

The term "bioactive" as used herein, refers to the activity or characteristic associated with the peptide and/or protein of interest. The bioactivity may be selected from enzymatic, molecular binding, elemental binding, antibacterial, antifungal, antiviral, antitumour, anti-inflammatory, or immunoregulatory properties.

The term "amino acid" refers to the basic chemical structural unit of a protein or polypeptide. The following abbreviations are used herein to identify specific amino acids:

As used herein, the term "chromatography" refers to a process in which a chemical mixture carried by a liquid or gas is separated into components as a result of differential distribution of the chemical entities as they flow around or over a stationary liquid or solid phase.

As used herein, the term "liquid chromatography" or "LC" means a process of selective retardation of one or more components of a fluid solution as the fluid uniformly percolates through a column of a finely divided substance, or through capillary passageways. The retardation results from the distribution of the components of the mixtu re between one or more stationary phases and the bulk fluid, (i.e., mobile phase), as this fluid moves relative to the stationary phase(s). Examples of "liquid chromatography" include reverse phase liquid chromatography (RPLC), high performance liquid chromatography (HPLC), reverse phase high performance liquid chromatograph (RP-HPLC) and high turbulence liquid chromatography (HTLC).

As used herein, the term "high performance liquid chromatography" or "HPLC" refers to liquid chromatography in which the degree of separation is increased by forcing the mobile phase under pressure through a stationary phase on a support matrix, typically a densely packed column.

As used herein, the term "mass spectrometry" or "MS" refers to an analytical technique to identify compounds by their mass. MS refers to methods of filtering, detecting, and measuring ions based on their mass-to-charge ratio, or "m/z". MS technology generally includes (1) ionizing the compounds to form charged compounds; and (2) detecting the molecular weight of the charged compounds and calculating a mass-to-charge ratio. The compounds may be ionized and detected by any suitable means. A "mass spectrometer" generally includes an ionizer and an ion detector. In general, one or more molecules of interest are ionized, and the ions are subsequently introduced into a mass spectrographic instrument where, due to a combination of magnetic and electric fields, the ions follow a path in space that is dependent upon mass ("m") and charge ("z").

As used herein, the term "operating in positive ion mode" as used herein, refers to those mass spectrometry methods where positive ions are generated and detected . The term "operating in negative ion mode" refers to those mass spectrometry methods where negative ions are generated and detected.

As used herein, the term "ionization" or "ionizing" refers to the process of generating an analyte ion having a net electrical charge equal to one or more electron units. Negative ions are those having a net negative charge of one or more electron units, while positive ions are those having a net positive charge of one or more electron units.

As used herein, the term "electrospray ionization" or "ESI," refers to methods in which a solution is passed along a short length of capillary tube, to the end of which is applied a high positive or negative electric potential. Solution reaching the end of the tube is vaporized (nebulized) into a jet or spray of very small droplets of solution in solvent vapor. This mist of droplets flows through an evaporation chamber. As the droplets get smaller the electrical surface charge density increases until such time that the natural repulsion between like charges causes ions as well as neutral molecules to be released.

The term "detecting" as used herein encompasses quantitative and/or qualitative detection of the target protein of interest. It will be appreciated that multiple reaction monitoring (MRM), selected reaction monitoring (SRM), consecutive reaction monitoring (CRM), and parallel reaction monitoring (PRM), are modes of analysis of a mass spectrometer.

Single/selected reaction monitoring (SRM) analysis utilizes a mass spectrometer (e.g. a triple quadrupole type of instrument) to select and analyse a specific analyte (such as a peptide or a small molecule). Selected reaction monitoring (SRM) is a method used in tandem mass spectrometry in which an ion of a particular mass is selected in the first stage of a tandem mass spectrometer and an ion product of a fragmentation reaction of the precursor ion is selected in the second mass spectrometer stage for detection. Following ionization in an electrospray source, a peptide precursor/parent is first isolated to obtain a substantial ion population of mostly the intended species. This population is then fragmented to yield product/daughter ions whose signal abundances are indicative of the abundance of the peptide in the sample. These peptide monitoring experiments are primarily performed on triple quadrupole mass spectrometers, where mass-resolving Q1 isolates the precursor, Q2 acts as a collision cell, and mass-resolving Q3 is cycled through the product ions which are detected upon exiting the last quadrupole by an electron multiplier. A precursor/product pair is often referred to as a transition. Much work is involved in ensuring that transitions are selected that have maximum specificity. By spiking in heavy -labelled (e.g., D, 13 C, or 15 N) peptides to a complex matrix as concentration standards, SRM can be used to construct a calibration curve that can provide the absolute quantitation of the target peptide fragment, and by extension, its parent protein.

It will be appreciated where multiple parent ions are monitored in a single MS run, this type of analysis is known as multiple reaction monitoring (MRM). Using MRM analysis, multiple proteins and multiple regions (signal peptides) of a protein can be monitored in single MS run. Consecutive reaction monitoring (CRM) is the serial application of three or more stages of selected reaction monitoring. Parallel reaction monitoring (PRM) is the application of selected reaction monitoring with parallel detection of all transitions in a single analysis using a high- resolution mass spectrometer. More generally, MRM measurement mode is a mode of MS / MS analysis in a tandem quadrupole mass spectrometer. In MRM measurement mode, the mass-to-charge ratio of ions that can pass through a quadrupole mass filter and a subsequent quadrupole mass filter are fixed respectively, to measure the intensity (quantity) of a specific product/daughter ion for a particular precursor/parent ion.

Assays

Proteins may be identified, screened for, or characterized by their physical/chemical properties and/or biological activities by various assays known in the art, such as the Bradford protein assay.

Assays can be used for identifying proteins having bioactive properties. Bioactive properties may include, e.g., antimicrobial activity.

Additional assays such as enzyme activity tests, molecular or elemental binding tests, and or antimicrobial activity tests may also be performed on a product to determine and confirm additional bioactive properties of the product.

One aspect of the invention provided herein is a reproduceable, efficient and/or economic LC- MS/MS-based method for quantification of at least two bioactive proteins in a sample, preferably a milk protein sample, such as a milk protein powder.

The invention generally relates to the use of mass spectrometry to identify and quantify peptide fragments of two or more proteins in a milk protein sample.

The Applicants previously identified in WO/2017/183996 (the entire contents of which are incorporated herein by reference) that combinations of bioactive proteins provided unexpected selectivity towards inhibition of pathogenic micro-organisms compared to a considerably less inhibitory effect towards beneficial commensal micro-organisms that are present in a healthy microbiome. A number of bioactive proteins were identified as being of interest, however it was unknown at the time how to identify and quantify these proteins in a sample in the efficient manner now detailed in this invention.

Following on from this work, the Applicant continued to conduct further research to identify as many proteins as possible in the cationic milk protein power and developed methodology to identify multiple target proteins in a sample. Initial studies identified over 58 proteins alone in the cationic fraction isolated from milk. The Applicant conducted further studies to identify and isolate specific target proteins of interest. The methodology is further discussed in the examples below.

During this work, a digestion method was utilised to identify unique peptide fragments that could be used to identify and potentially quantify the presence of proteins of particular interest. The identification of the unique peptide fragments provided the basis for developing a method according to the present invention using mass spectrometry, whereby MRM LC-MS/MS is a particularly preferred technique.

Preferred proteins of particular interest include lactoferrin (LTF), lactoperoxidase (LPO), Lysosomal alpha mannosidase (LAM), Immunoglobulin G (IgG), Ribonuclease (Rnase), and Angiogenin (Ang); some or all of which may be present in a mixture of milk proteins.

It is believed that the presence of two or more of these proteins in a product may provide a therapeutic effect to a product, in particular, the presence of lactoferrin and/or lactoperoxidase are especially believed to be beneficial and provide unique bioactive properties.

However, it is time consuming and difficult task to determine whether a particular source of milk protein will contain two or more these proteins and, if so, how active the proteins have remained following manufacture and how much of the proteins are included in the milk protein source. It is therefore of great interest to consumers and manufacturers alike to provide a quick and simple method to identify and quantify for the presence of two or more bioactive proteins in a product, such as a milk protein product. A single identified previous method for identifying lactoferrin in a milk powder sample was disclosed in Yuan et al., Selection of possible signature peptides for the detection of bovine lactoferrin in infant formulas by LC-MS/MS, PLOS one

(https://doi.org/10.1371/iournal.pone.0184152.t001). That reference discloses a LC-MS/MS assay based on a signature peptide for a single protein, lactoferrin, in infant formulas. Yuan et at. does not explore or attempt to identify or quantify any other additional proteins in the sample. Apart from determining the quantity of lactoferrin present in the infant formulas tested, no further identification or determination of the overall bioactive or therapeutic properties for each infant formula can be made. Furthermore, it has been determined that the specific peptide sequence used by Yuan et al. was not the most specific unique peptide sequence available for best determining the quantity of lactoferrin present. Perhaps the most significant shortcoming of Yuan et al. for determining the bioactivity of samples that contain milk proteins is that, of all individual cationic milk proteins, lactoferrin is one of the least bioactive. As a protein, its primary activity is simply binding iron and sequestering iron, which is its primary purpose in infant formulas. For any therapeutic applications requiring bioactivities, such as antimicrobial and/or anti-inflammatory and/or antioxidant activities, the identification of lactoferrin on its own would not be sufficient for assessing the contribution of the milk protein product to the therapeutic bioactivity contribution of the sample.

In contrast, the methodology of the present invention provides for the quick and efficient quantification of two or more, or more generally three or more, or generally four or more, or particularly five or more, or more particularly six or more bioactive proteins of interest in a single test and provides a useful measure for determining whether a particular milk protein product contains beneficial bioactive properties. In addition, it also allows for the identification and determination of the overall beneficial therapeutic advantages of the product in comparison with other competing products.

Based on the inventors' identification of specific, unique peptide fragments associated with the proteins of interest the inventors have devised an efficient and cost-effective method by which those specific, unique peptide fragments may be identified and quantified within a sample that contains milk protein.

The unique peptide fragments may be provided by the treatment of a milk protein sample with a digestive enzyme, such as trypsin. The function of the digestive enzyme is to cleave the protein of interest into peptide fragments which can then be analysed by mass spectrometry.

The present methods, utilising mass spectrometry, allow for a large number of milk proteins to be analysed in a quick and efficient manner from a single sample, in order to identify and quantify the peptide fragments of interest and thereby identify and quantify the proteins themselves.

The methods devised by the inventors address the issues of determining whether a given milk protein sample or milk product contains any bioactive or therapeutic effect, and also provides for the determination of the likely magnitude of any bioactive/therapeutic effect. It can also at least identify the magnitude of a bioactive property in at least one or more the milk proteins present in the sample. The results can be applied to milk protein-based products to provide a useful measure for consumers or industry to determine the overall beneficial properties between competing products.

It is envisaged that results of the analysis can be utilised in an index rating system, with a 'score' or rank being applied to the products tested and provide a meaningful measure for a consumer or manufacturer to compare products.

It will be appreciated that similar index/rating systems already exist in the industry for consumers or manufacturers to distinguish products based on inherent therapeutic benefits from one another. By way of example, there are currently two rating systems being utilised for Manuka Honey. It is now believed a similar indexing system can be applied to milk protein products as tested with the methodology of the present invention. Such an indexing system may be used to standardise milk protein powders or products containing milk protein powders. In addition, further additional testing can be performed on the milk protein product to establish any further likely bioactive/therapeutic properties present in the product or at least identify the magnitude of a bioactive property in at least one or more the milk proteins present in the sample, the results of which can be added or combined with the initial analysis discussed above to determine an overall rating for the product.

Examples of additional testing include, but not necessarily limited to: activity testing, such as specific enzyme activity testing, antimicrobial testing, anti-inflammatory testing or the like.

In particular, preferred additional testing may include a lactoperoxidase activity test and/or an antimicrobial activity assessment. The tests can either be conducted in the alternative or in combination with one another.

As an example, the lactoperoxidase activity test can be used to check if lactoperoxidase is present or has been added to a product at the appropriate or claimed dose. It can be further be used to test the stability of lactoperoxidase in the product.

In addition, the antimicrobial activity assessment can be used to determine the level of antimicrobial activity of a particular protein and/or the milk protein product in vitro. Examples include gram-positive or gram-negative organisms such as Klebsiella aerogenes (such as Klebsiella aerogenes NZMR 798 (ATCC 13048 / DSM 30053), E. coli, S. aureus, C. albicans, S. epidermidis, S. mitis, S. mutans and S. salivarius.

The inventor has found that the use of Klebsiella aerogenes NZMR 798 (ATCC 13048 / DSM 30053) in assessing antimicrobial activity is superior to the use of E.coli. It has been found that K. aerogenes is more sensitive to milk proteins than E. coli, and this greater sensitivity allows for differentiation of smaller differences between samples. A strain of Cronobacter sakazakii was also tested as it was thought to be appropriate as it has been isolated from infant formula. However, it was less sensitive to milk proteins than K.

aerogenes.

Without wishing to be bound by theory, it is believed that in general the more sensitive the bacteria is to the milk proteins the better insofar as being able to readily assay activity and gain results. Preferably the bacterial species used in the antimicrobial activity assessment is sensitive to the milk proteins at concentrations of at least 0.3 mg/mL.

The bacteriostatic and bactericidal properties of milk proteins are likely to be the result of multiple mechanisms. Different species and strains of bacteria respond differently to each mechanism. For example, lactoferrin, one of the proteins found in a desired cationic milk fraction binds iron. As such it is believed that bacterial strains with high iron demands will be inhibited more by lactoferrin than bacterial strains with lower iron demands. It is believed that K. aerogenes is more sensitive to one or more of the bacteriostatic and or bactericidal mechanisms of the fraction than E. coli resulting in its greater sensitivity to the proteins.

The initial scoring method (A) was based on the number of wells in which growth had been inhibited (which was determined as not exceeding 15% of the growth in the control well after 18 hrs). Each product was tested in triplicate at eight concentrations. The milk fraction was tested against two bacterial strains (K. aerogenes and S. aureus NZMR 87). Eight concentrations times three replicates times two strains gave a maximum possible score of 48. Product was tested at eight concentrations times three replicates against S. aureus. The result was multiplied by two to give a score of 48 to make it comparable with the IDP test on a 'per dose' basis. This is mainly due to the fact that, unlike indexing in honey, where 100% of the product is one substance, active milk proteins are powerful enough that they are added as much smaller components of a finished product. Therefore, indexing on a 'per dose' basis is important for consumers to understand the true power of the product on a practical day-to-day level.

Example of scoring method A for a cationic milk fraction: A 96 well plate was set up as shown in Figure 13. As only the wells in row H of the fraction K. aerogenes dilutions exceeded 15% of the growth in the control wells it was given a score of 7. As only the wells in row H of the fraction S. aureus dilutions exceeded 15% of the growth in the control wells it was given a score of 7. The process was repeated two more times with the same result. The fraction scored 7 for each of the bacteria and for each of the three replicates, giving a per dose score of 42 out of a possible 48.

Example of scoring method A for product:

A 96 well plate was set up as shown in Figure 14. As no growth was observed in the first five rows of the product 1 dilutions it was given a score of 5. The process was repeated two more times with the same result. The product scored 5 for each of the three replicates, giving a score of 15. This was multiplied by 2 to give a per dose score of 30 out of a possible 48.

Scoring using the initial method (method A) did not reflect a reduction of bacterial growth. To assign a score that reflected this, a second scoring method based on percent reduction in growth, in comparison to growth in the media only controls, was developed. Percentage reduction in growth, at three concentrations, over three replicates is calculated, and the result divided by 18 to give a score out of 50.

Example of scoring method B for product:

Using the data from the 96 well plate shown in Figure 14 the average growth in the media + NaSCN +ve control wells was calculated using the formula:

C = (A1@14h-A1@20min + B1@14h- B1@20min + C1@04h- C1@20min) ÷ 3

In this case, C = (0.397-0.076 + 0.374-0.075 +0.4-0.075)/3 = 0.315

For each sample the average growth for rows B, E, and F is calculated, as a %, using the following formula :

GR = 100 ÷ C x ((R4@ 14h-R4@20min + R5@14h-R5@20min + R6@14h-R6@20min) ÷ 3)

Where R = the row. For row H:

GH = 100 ÷ 0.315 x ((0.396-0.078 + 0.352-0.077 + 0.353-0.076) ÷ 3) = 92

This is repeated for rows B, E, and F in each replicate. Occasionally a slight drift in reading during incubation results in a small negative GR score. This is treated as a zero score.

The average growth for each row is converted to average reduction in growth:

ER = 100 - GR

For row FI:

EH = 100 - 92 = 8

Again this is repeated for rows B, E, and F in each replicate.

The results from each of three rows from each of the three replicates are ad ded up and divided by 18 to give a maximum possible score of 50.

Figure 14 score = (100 + 100 + 8 + 100 + 100 + 19 + 100 + 100 +11)/18 = 35

Note: the second reading is used as the zero value because the value changes slightly in the first

20 minutes as the plate reaches 37 °C.

A second advantage scoring method B is that if a sample achieves the maximum score of 50 a further dilution can be made. The score can them be calculated at four concentrations divided by 18 giving the possibility of a score up to 67. Further dilutions could be added if needed. This avoids a 'ceiling effect' or maximum achievable score.

Using the data from the 96 well plate shown in Figure 15 the average growth in the media + NaSCN +ve K. aerogenes control wells was calculated using the formula :

C = (A1@14h-A1@20min + B1@14h- B1@20min + C1@14h- C1@20min) ÷ 3

C = (0.518-0.075 + 0.522-0.076 + 0.618-0.075)/3 = 0.477

For each sample the average growth for rows B, E, and F is calculated, as a %, using the following formula :

GR = 100 ÷ C x ((R4@14h-R4(5 ) 20min + R5@14h-R5@20min + R6@14h-R6@20min) ÷ 3)

Where R = the row.

For row H: GH = 100 ÷ 0.477 x ((0.561-0.076 + 0.486-0.075 + 0.491-0.074) ÷ 3) = 92

This is repeated for rows B, E, and F in each replicate. Occasionally a slight drift in read ing during incubation results in a small negative GR score. This is treated as a zero score.

The average growth for each row is converted to average reduction in growth:

ER = 100 - GR

For row H:

EH = 100 - 92 = 8

Again this is repeated for rows B, E, and F in each replicate.

The same calculations are carried out using the S. aureus data.

The results from each of three rows from each of the three replicates from the two bacterial strains are added up and divided by 32 to give a maximum possible score of 50.

Figure 15 Score = (8+28+29+9+23+28+6+17+21+11+17+18+4+4+20+16+ 14+29)/36 = 8

The results of the additional testing can be weighted depending on the results obtained for each milk protein product to provide further consideration to the overall index rating for the product.

It will be appreciated that any number or combination of additional tests may be performed on the milk protein product and provided for consideration to the overall index rating system.

It will also be appreciated that the method for indexing can be normalised to a 'dose' of a milk protein product as opposed to the entire product as a whole. For example, it will be readily understood that milk protein-containing products may include other components and additives, resulting in a final product not being 100% milk protein. Since the milk protein is inevitably the most expensive component of any milk protein product, manufacturers can include the milk protein, but fail to specify or quantify how much is in the product. Therefore, indexing this amount according to the dose of the product is an important measure and removes ambiguity to the consumer and/or industry.

According to a further aspect of the invention, the method uses mass spectrometry to determine and quantify the presence of two or more peptide fragments in a milk derived protein sample and thereby identify and quantify their respective proteins.

The method preferably uses multiple reaction monitoring (M RM) in a liquid chromatography tandem-mass spectrometry (LC-MS/MS) method .

As previously discussed, MRM is a measurement mode of MS / MS analysis in tandem quadrupole mass spectrometer. In MRM measurement mode, the mass-to-charge ratio of ions that pass through a quadrupole mass filter and a subsequent quadrupole mass filter are identified and set respectively, to measure the intensity (quantity) of specific product/daughter ions for a particular precursor/parent ion associated with the target protein.

In order to perform high accuracy quantification of the target protein using MRM, it is necessary to identify and define appropriate measurement parameters according to the target protein. The MRM measurement parameters include mass-to-charge ratio of the

precursor/parent ion mass to charge ratio of product/daughter ions, collision energy (voltage), which all need to be determined for the protein of interest.

To enable the determination and quantification of two or more proteins in a sample, the inventors have spent considerable time and have identified the unique peptide fragments for the proteins of interest.

In particular, the mass to charge ratio (m/z) for the parent ions of the unique peptide fragments identified by the inventors are:

Additionally, the inventors have also identified the following product/daughter ions from the proteins of interest with the following mass/charge ratios:

wherein QT; quantifying ion, QF; qualifying ion.

Using two or more of the identified peptide fragments, the inventors have devised an efficient and cost-effective method by which two or more target proteins may be identified and quantified within a sample of milk protein.

The method provides for analysis of a base product or processed milk powder for bioactive proteins with minimal sample preparation using multiple reaction monitoring (MRM) liquid chromatography tandem-mass spectrometry (LC-MS/MS).

In general, the method is applicable to powder and/or liquid milk products and requires minimal sample preparation prior to undergoing quantification by mass spectrometry.

In one embodiment, the method can be used to analyse milk protein powders, including cationic protein powders.

In particular, the method comprises the following steps: the sample is reconstituted (if in powdered form), before undergoing proteolytic digestion with a digestive enzyme such as trypsin in a suspension containing known amount(s) of heavily labelled peptide(s) that act as an internal standard (ISTD). Calibration standards may also be prepared and run with the samples for validation.

It will be appreciated that the preparation of the samples for analysis with the method of the present invention provides for a much quicker and efficient work up than required for previous analytical methods. Sample preparation for the present method does not require any additional assays to separate out the proteins of interest nor require any substantial purification methods (typically a liquid chromatography technique will be used immediately prior to the

fragmentation of the protein fragments in the mass spectrometer. This leads to vast improvements in efficiency of testing, increased volumes of samples that can be tested and also quicker turnaround times to report results.

Extracts are preferably analysed on a LC-MS/MS apparatus such as UPLC-MS/MS with a C18 Hypersil GOLD column together with a series of calibration standards containing the same amount of ISTD. Unique identification is preferably performed on a triple-quad mass spectrometer (QQQ) by detecting the specific mass of target peptide ions and quantifying specific product/parent ions at optimised collision energies. The concentration of proteins of interest present in the test samples may be determined from the calibration curve provided by the calibration standards, and the relative abundance of the parent protein can then be calculated accordingly.

The bioactive proteins that can be quantified by the method of the present invention include: lactoferrin (LTF), lactoperoxidase (LPO), Lysosomal alpha mannosidase (LAM), Immunoglobulin G (IgG), Ribonuclease (Rnase), and Angiogenin (Ang).

The unique peptide ion fragments

The following examples serve to illustrate the invention. These examples are in no way intended to limit the scope of the methods.

All references, including any patents or patent applications cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents form part of the common general knowledge in the art.

It is acknowledged that the term 'comprise' may, under varying jurisdictions, be attributed with either an exclusive or an inclusive meaning. For the purpose of this specification, and unless otherwise noted, the term 'comprise' shall have an inclusive meaning - i.e. that it will be taken to mean an inclusion of not only the listed components it directly references, but also other non-specified components or elements. This rationale will also be used when the term

'comprised' or 'comprising' is used in relation to one or more steps in a method or process.

EXAMPLES/METHODOLOGY

EXAMPLE 1 - Determination of bioactive proteins in a product sample

Initial studies were conducted by the inventors to identify and separate the proteins of interest from a milk protein sample.

Preparation of test samples

Test samples were solubilised to enable the protein concentration of each sample to be determined. Three different product samples were analysed according to a method as follows. Each product sample were tested at a concentration of 25mg/g of sample and include:

• A milk protein powder;

• Strawberry flavoured tablet;

• Mint flavoured lozenge.

800 mg of the milk protein sample was dissolved in 8.0 mL of type-1 water to provide a 10% (w/v) solution.

The strawberry tablet was crushed into a fine powder using a pestle and mortar with a total of 800 mg being dissolved in 8.0 mL of type-1 water to provide a 10% (w/v) solution.

The mint lozenge was also crushed into a fine powder using a pestle and mortar with a total of 800 mg was dissolved in 4.0 mL of type-1 water to provide a 20% (w/v) solution. The samples are vortexed briefly to ensure mixing and the lids wrapped in parafilm to prevent leakage and then placed on a rotary mixer for 30 minutes.

The suspensions are then sonicated for approximately 10 minutes to aid protein solubilisation and then mixed again for a further 30 min on a rotary mixer. 1.6 mL of the suspensions are then transferred to a 2.0 mL micro-centrifuge tube and the centrifuged for a further minute at 12,000 x g to pellet any undissolved material. A 1.0 mL of supernatant is then collected and transferred to a new micro-centrifuge tube for further analysis.

Following this preparation, the samples are subjected to Bradford protein assay to determine the total protein concentration in each solution.

Bradford Protein assay:

The Bradford protein assays were used to measure the concentration of total protein in the sample. The principle of the assay is that the binding of protein molecules to Coomassie dye under acidic conditions results in a colour change from brown to blue which can be measured using a spectrophotometer.

40mL of Bradford reagent concentrate 1 in 5 was diluted with 160 mL Type-1 water. This solution was filter through Whatman #1 filter paper to provide the working solution of the Bradford reagent.

2 mg/mL albumin standard (BSA) two-fold was diluted in Type-1 water to make a 1 mg/mL BSA standard solution.

Six dilutions of protein standard were prepared using 1 mg/mL BSA with a range of 3 to 18 pg protein. The volume of these standards were made up to 100 mL with Type-1 water (Table 1).

Table 1: BSA standard samples for Bradford assay calibration curve

The protein samples were diluted 10-fold in Type-1 water and subsequently mixed (i.e. 100 mL + 900 mL).

A blank tube for the standard curve was prepared with 100 mL of Type-1 water (0 mg/mL). 15 mL of each milk protein powder and Strawberry tablet sample was separately diluted with 85 mL with Type-1 water. While 30 mI_ of the mint lozenge sample was diluted with 70 mL of Type-1 water.

900 mL of diluted Bradford solution was then added to each micro-centrifuge tube and mixed well followed by incubation at room temperature for at least 5 minutes. As absorbance increases over time, the samples are measured within 30 min and were not incubated for a period longer than 1 hour.

A l mL aliquot of each standard protein mix is then transferred to a plastic cuvette and the absorbance at 595 nm is measured.

The water blank sample provided for the standard curve is then measured. The assay is zeroed to the water blank and then measured against each protein standard to generate the 0-18 mg/mL standard curve.

The unknown protein samples are then measured against the standard curve to determine the protein concentrations for each sample using a quadratic formula (see below). Alternatively, 200 mL can be transferred to a well of a 96-well microplate and the absorbance measured at 595 nm.

Worksheets used for performing analytical quadratic calibration curves can be found at the following web address: https://terpconnect.umd.edu/~toh/models/CalibrationCurve.htm l

The protein concentration of original supernatant is calculated considering the dilutions and volumes used for the Bradford assay. The expected protein concentrations for the three samples are shown below in Table 2:

Table 2: Expected protein concentration range from the samples

SDS-polyacrylamide pel electrophoresis ( SDS-PAGE ) analysis

The separation of macromolecules in an electric field (electrophoresis) was then undertaken. A very common method for separating proteins by electrophoresis uses a discontinuous polyacrylamide gel as a support medium and sodium dodecyl sulphate (SDS) to denature the proteins and provide a consistent charge/mass ratio. The method is called sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Protein separation by SDS-PAGE can be used to estimate relative molecular mass, to determine the relative abundance of major proteins in a sample, and to determine the distribution of proteins among fractions. The purity of protein samples can then be assessed, and the progress of a fractionation or purification procedure can be followed thereafter.

The following buffers were used in the analysis: 1M Tris, pH 6.8 (500 mL):

To 60.57 g Tris base, 400 mL of Type-1 water was added. Add concentrated HCI to bring pH to 6.8 and make up to 500 mL with additional Type-1 water.

SDS sample buffer (3x):

Glycerol (30% v/v)

b-mercaptoethanol (7.5% v/v)

SDS (6% w/v)

Tris pH 6.8 (187.5 mM) MES running buffer:

Dilute 50 mL 20x MES buffer into 950 mL Type-1 water to make lx M ES running buffer

Fixing solution (1 litre): 500 mL Methanol (50% v/v)

20 mL Phosphoric acid (2.0% v/v)

Make to 1 litre with Type-1 water

Colloidal Coomassie Blue stain (1 litre):

170 g Ammonium sulphate (17% w/v)

20 mL Phosphoric acid (2.0% v/v)

340 mL Methanol (34% v/v) (add very slowly with continuous mixing)

0.6 g Coomassie Blue G-250 (0.06% w/v)

Dissolve ammonium sulphate in 400ml Type-1 water. Add 20 mL phosphoric acid and make up to 660 mL with Milli-Q water. Slowly add 340 mL of methanol. Dissolve 0.6 g of Coomassie G- 250 in a few mL of the methanol. Add this to the 1 litre solution. Filter the solution if you are concerned about dye solubility.

Destain solution (1 litre): 5% (v/v) methanol

50 mL Methanol

Make to 1 litre with 950 mL Type-1 water.

The milk protein powder and strawberry tablet protein samples were made up to 3 mg/mL with 3x SDS sample buffer, while the mint protein sample was made up to 1.5 mg/mL with 3x SDS sample buffer. The samples were subsequently boiled for 2 min, vortexed and pulse spun.

A pre-cast 12% Bis-Tris gel was placed into a Criterion transfer tank and lx M ES running buffer was added following the manufacturer's instructions. 5 mL of SeeBlue Plus2 molecular weight (Mwt) standard is loaded to an outside well.

Using gel loading tips, 20 mL of the milk protein sample (60 pg), strawberry sample (60 pg), and mint sample (30 pg) sample are loaded individual wells. 10 mL of 3x SDS sample buffer was then loaded to wells not containing any Mwt or protein sample. The protein samples were then subjected to electrophoresis at 80 V (constant voltage) for the first hour and then subsequently increased to 150 V until the dye front was approximately 1 cm from the bottom of the gel. Once electrophoresis was completed, the gel was removed from the plastic support and placed into 150 mL of a fixing solution.

The gel was washed with three changes of fixing solution for 30 min each with slight agitation to fix the proteins onto the gel but also to remove any excess SDS or other chemicals that may contaminate the Coomassie stain. 150 mL of colloidal Coomassie stain was then added and allowed to stain overnight. Colloidal Coomassie stains the protein but has minimal staining of the gel allowing for easy destaining.

Once the time has elapsed, the stain was removed, and the gel was washed with several changes of destain solution until the background was clear of stain.

The gel was then scanned using a calibrated densitometer or similar scanner as shown in figure 1

In-gel tryptic digestion

Tryptic digestion was then used to digest the protein bands into smaller peptide fragments for mass spectrometry, particularly tandem mass spectrometry identification.

The following buffers were used in the analysis.

Digestion buffers:

iv Wash buffer (10 mL): 50% (v/v) acetonitrile/25 mM ABC

To 5 mL of acetonitrile add 500 mL of 500 mM ABC and 4.5 mL of type-1 water.

v 25 mM ABC (4 mL):

Dilute 500 mM ABC 1 in 20. i.e. 100 mL of 500 mM ABC + 1900 mL of type-1 water. vi lO mM DTT (l mL): lO mM DTT/25 mM ABC

To 930 mL of type-1 water add 20 mL of 500 mM DTT and 50 mL of 500 mM ABC.

vii 55 mM IAA (1.5 mL): 55 mM IAA/25 mM ABC

To 1260 mL of type-1 water add 165 mL of 500 mM IAA and 75 mL of 500 mM ABC. viii Digest buffer (2 mL): 5% (v/v) acetonitrile/25 mM ABC

To 1800 mL of type-1 water add 100 mL of acetonitrile and 100 mL of 500 mM ABC. ix Trypsin solution: (0.1 mg/mL):

Add 200 mL of digest buffer to a glass vial containing 20 pg of sequencing grade trypsin. x Extraction buffer (4 mL): 40% (v/v) acetonitrile/0.2% formic acid

To 1.6 mL of acetonitrile add 9 mL of 90% formic acid and 2391 mL of type-1 water.

Dav-1: Band selection and destaining

Fresh stock solutions of 500 mM ABC, 500 mM DTT and 500 mM IAA were made up as well as wash buffer (50% acetonitrile/25 mM ABC).

Four gel slices were cut out from each lane for analysis with each gel slice being cut into slivers to increase available surface area (see figure 2). Gel slivers from each individual gel slice, from each lane, were then placed into four individual 1.5 mL eppendorf Lo-Bind tubes to provide 12 tubes in total - one for each gel slice.

The gel slices were then destained with 200 mL of wash buffer. A least three washes were performed to remove the Coomassie stain from the gel slices. The first wash was used to remove most of the stain and incubated for at least 2 hours. After the second wash the gel slices were incubated overnight at 4 °C. The third wash was used to clean up any residual stain the following morning (1-2 hours). Dav-2: Tryptic digestion

The gel slices were then dehydrated with 100% acetonitrile and speed-vac to dryness for 10-15 minutes. 80 mL of 10 mM DTT solution was then added to reduce any cysteine residues, followed by incubation at 37 °C for 30 minutes.

The slices were allowed to cool to room temperature, with any residual DTT liquid being pipetted off with a gel-loading tip. 120 mL of freshly prepared 55 mM IAA solution was then added. The proteins were alkylated for 2 hours at room temperature in the dark.

The liquid is pipetted off and the gel pieces were washed with 200 mL of 25mM ABC to remove any residual IAA and left for at least 30 minutes.

The 25 mM ABC was removed and the gel pieces were then dehydrated for 15 minutes with 100 mL wash buffer and then once more for 5 min with 200 mL of 100% acetonitrile. The digest buffer was then made up, followed by the 0.1 pg/mL trypsin solution in the digest buffer.

The gel pieces were then rehydrated for 30 minutes on ice in 20 mL of trypsin solution. The rehydrated gel pieces are overlaid with 100 mL of digest buffer to keep them immersed throughout the digestion process and left to incubate overnight at 37 °C (16-24 hour).

Note: that should the volume of buffer vary depending on the size of the gel pieces, then more buffer was added if necessary to immerse the gel pieces.

Day-3: Peptide recovery

The digests were pulse-spun and sonicated for 10 minutes. Extraction buffer (40%

acetonitrile/0.2% formic acid) was then prepared with the digest solution (DS) being transferred to a new 0.6 mL Lo-Bind tube and kept. 120 mL of extraction buffer was added to the remaining gel pieces and sonicated for a further 10 minutes followed by pulse-spinning. The previous step was repeated with the supernatants (DS) for each gel piece being pooled together. 60 mL of 100% acetonitrile was then added to the gel pieces and left for 2 min before the supernatant was transferred to the rest of the pooled supernatants (DS).

Each individual tube was found to approximately contains 400 mL of peptide digest (DS). The samples were then pulse spun and place in -80 °C freezer for 30 minutes to freeze, followed by speed-vac to dryness for ~ 2 to 3 hours.

The samples are now ready for LC-MS/MS analysis.

Detection/analysis by LC-MS/MS

Mass spectrometry (MS) is an analytical technique that ionizes chemical species and sorts the ions based on their mass to charge ratio. Tandem mass spectrometry, also known as MS/MS or MS 2 , involves multiple steps of mass spectrometry selection, with some form of fragmentation occurring in between the stages.

The separation and identification of peptides in a complex mixture first requires

chromatographic separation on a column containing a solid hydrophobic support stationary phase. This is known as reversed-phase high performance liquid chromatography (RP-HPLC). Peptides are sequentially released from the solid support by a gradient of mobile phase usually containing a mixture of aqueous buffer and organic solvent such as acetonitrile.

The use of a mass spectrometer and interpretation of the data requires expert knowledge in their operation. Accordingly, the following contains relevant information to duplicate the conditions used to separate, fragment and identify the target peptides and proteins.

An Ultimate 3000 LC system was used for the separation of the protein digests. Buffer A (0.2% formic acid in water) and buffer B (0.2% formic acid in acetonitrile) were used as mobile phases for gradient separation. For each run the protein digests from each gel slice were loaded onto a Hypersil Gold C18 column at 3% buffer B with a flow rate of 0.6 mL/min at the volumes listed below in Table 3. The column temperature was maintained at 55 °C. Bound peptides were eluted using a linear gradient separation listed below in Table 4.

Peptides eluted from the column were analysed in data-dependent MS/MS mode on a qExactive Orbitrap mass spectrometer using a heated electrospray ionization (HESI-II) source for ionization in positive ion mode. The instrument parameters were as follows: the capillary temperature was set to 350 °C, and the electrospray voltage was 4.0 kV. The MS instrument was operated in a top 5 data-dependent acquisition (DDA) mode to automatically switch between full-scan MS and MS/MS acquisition. Fullscan MS spectra (m/z 200-2000) were acquired in the Orbitrap with resolution 70,000 (at m/z 200). Automatic gain control (AGC) target value was 1 xl06 counts, and the maximum injection time was 100 ms.

For tandem MS spectra, the five most abundant precursor ions with charge state >2 were fragmented in the HCD collision cell, using an isolation width of 4.0 m/z, a normalized collision energy of 30%, and a mass resolution of 17,500 (at m/z 200). The ion selection threshold was 1 x 105 counts, and the maximum allowed ion accumulation time was 50 ms. For all analyses, the dynamic exclusion time was set to 15 s.

Table 3: Injection volumes for protein digests

For each sample, 15 mL for Slices-1 and -2 were automatically loaded and 10 mL for Slices-3, and -4 were automatically loaded. Table 4: Reverse phase elution gradient for MS/MS analysis

Data analysis

Raw data files for each gel slice were then imported into PEAKS Studio v7.5 (Waterloo, ON, Canada). Gel slices for each protein sample were searched together (n = 4) against an 'in-house' database containing the proteins of interest (Table 5). Searching the four gel slices together improves detection of proteins that may be present in more than one gel slice (i.e. LAM which can be observed in slices 1 and 2) and to limit the output to one result file.

Database searching parameters included up to two missed cleavages for semi-tryptic digestion, precursor ion mass tolerance of 10 ppm, with a product ion mass tolerance of 0.1 Da. Cysteine carbamidomethylation was set as a fixed modification, and oxidised methionine (M), phosphorylated (STY) and deamidated (NQ) were set as variable modifications. The peptide false discovery rate (FDR) was estimated by the decoy fusion method and was set to a maximum of 5%.

Using these settings two or more unique peptides were required to positively identify each target protein.

Table 5: Target proteins

* Immunoglobulin gamma heavy chain is not found as an entry in the UniProt Swiss-Prot database. A partial sequence for this protein can be found at the National Center for Biotechnology Information using the NCBI accession number ABE68619.

Alternative peptide identification method

Lysosomal alpha-mannosidase (LAM) was found to be the least abundant of the six target proteins in the milk protein powder and tablet products.

This presented some difficulties in its detection. From the samples tested, protein loadings of less than 60 pg on a ID gel may result in LAM not being detected in Slices-1 and -2 using the automated database searching algorithm. The inventors believe there are two possible reasons for this; firstly, there was a relatively high abundance of additional milk proteins (caseins and whey proteins) found in the milk protein powder and strawberry tablet which decreased the amount of LAM loaded onto the gel lane. Secondly, the high abundance of lactoferrin and lactoperoxidase in Slices-1 and -2 may also contribute with interfere in the detection of LAM peptides. Accordingly, a 5% FDR threshold was employed to minimise this effect.

Alternatively, a more targeted approach could also be used to identify peptide ions specific to LAM. By using the accurate mass of known LAM tryptic peptides, these precursor peptides can be singled out amongst the raw MS spectrum of Slice-1 and Slice-2 using the Qual Browser in Xcalibur 4.0 software (Thermo Scientific) or similar programme.

A list of five targeted peptides specific to LAM and their observed mass-to-charge ratios were identified and listed below in Table 6. A scan-based plot of the five targeted ions identified in Slice-1 of the milk protein powder is provided as an example in Figure 3. All five target ions are easily identified from the chromatogram as single peaks eluting off the column during the LC- MS/MS run.

Table 6: Targeted peptides specific to Lysosomal alpha-mannosidase (LAM).

EXAMPLE 2 - Selection of signature peptides for detection by LC-MS/MS MRM analysis With the identification of the target proteins, the inventors proceeded to establish

methodology for their identification and quantification using LC-MS/MS MRM analysis.

One of the most critical steps for establishing LC-MS/MS approaches for protein quantitation is selecting suitable signature peptides from the tryptic hydrolysates. While the use of computational programs can predict tryptic cleavage sites, they do not reveal how the peptides will perform on-column. Alternatively, tryptic peptides identified by LC-MS/MS analysis provide information on the abundance of their ions and chromatographic performance under similar conditions to the MRM assay.

Candidate peptides are selected based on several critical factors:

i Specificity to the target protein (BLAST search)

ii Size of the peptide (7 to 16 amino acids long)

iii Water solubility and hydrophobicity

iv Chromatographic performance

v Reproducibility between sample preparations

vi The relative intensity of the MS signal

vii They should not contain amino acids susceptible to chemical modification such as

cysteine and methionine.

viii They should not contain specific motif sites (glycosylation and phosphorylation) ix No proline residue after tryptic cleavage site

x Ideally, the LC-MS/MS data should ideally identify a fully tryptic peptide without

multiple non-specific cleavages surrounding it. (see examples of PEAKS data below)

Finally, narrow peak shape, good signal intensity, no crosstalk (in transitions) between other peptides and acceptable elution times were additional key criteria that guided the final peptide selection.

The following is a concise description of the peptide selection process. Lactoferrin:

In silico tryptic digestion of lactoferrin (P24627) using Peptide Mass (https://web.expasy.org) identified 32 peptides, 7 to 16 amino acids in length. Elimination of peptides containing either cysteine or methionine residues reduced the total to 13 peptides. Four of these peptides contained glutamine residues (Q), which are prone to deamination, leaving a total of nine potential diagnostic peptides as shown in Table 7.

Table 7: Physical properties of lactoferrin in-silico tryptic peptides

The online BLAST search in UniProt (www.uniprot.org) and NCBI (www.ncbi.nhn.nih.gov) were used for evaluating the specificity of the selected signature peptides. Peptides unique to lactoferrin were chosen for further evaluation. To determine which peptide was best identified by tryptic digestion and LC-MS/MS analysis, a sample of co-isolate was digested, and the raw data analysed using PEAKS Studio v8.5. The PEAKS Studio software gives an indication of the total peptide intensity providing information on the most abundant diagnostic peptides. Peptides showing a larger area intensity could mean that the peptide ionises and chromatograms better than other peptides, or that the sequence is more accessible/receptive to the enzyme providing better enzyme efficiency. In many ways, this is the best method for determining the best peptide for AQUA analysis.

The peptide coverage map for the tryptic digestion of lactoferrin (SEQ ID No 1) is shown in Figure 4.

Peptide intensities were determined for the five unique peptides along with a non-specific peptide that showed a clean digest profile (SVDGKEDLIWK) with high peak area intensity. For some unknown reason, the lysine in the middle of the SVDGKEDLIWK sequence was not cleaved by trypsin. The co-isolate data is shown below in Table 8.

Table 8: Summary of tryptic peptides from co-isolate lactoferrin.

n.d. not detected

Both peptides ETTVFENLPEK and ANEGLTWNSLK contain an asparagine residue (N). This amino acid is also prone to deamination. Peptide VDSALYLGSR is of lower intensity than the other peptides.

Based on the above findings, the following signature peptides were selected for peptide synthesis:

LRPVAAEIYGTK (SEQ ID No 7)

SVDGKEDLIWK (SEQ ID No 8) Lactoperoxidase:

In silico tryptic digestion of lactoperoxidase (P80025) using the Peptide Mass programme (https://web.expasy.org) identified 29 peptides, 6 to 16 amino acids in length. Elimination of peptides containing either cysteine or methionine residues reduced the total to 15 peptides. Nine of these peptides contained glutamine residues (Q) which are prone to deamination, leaving a total of six potential diagnostic peptides as shown in Table 9.

Table 9: Physical properties of lactoperoxidase in-silico tryptic peptides

The online BLAST search in UniProt (www.uniprot.org) and NCBI (www.ncbi.nhn.nih.gov) were used for evaluating the specificity of the selected signature peptides. Peptides unique to lactoperoxidase were chosen for further evaluation. To determine which peptide was best identified by tryptic digestion and LC-MS/MS analysis, a sample of co-isolate was digested, and the raw data analysed using PEAKS Studio v8.5. The PEAKS Studio software gives an indication of the total peptide intensity providing information on the most abundant diagnostic peptides. Peptides showing a larger area intensity could mean that the peptide ionises and chromatograms better than other peptides, or that the sequence is more accessible/receptive to the enzyme providing better enzyme efficiency. In many ways, this is the best method for determining the best peptide for AQUA analysis. The peptide coverage map for the tryptic digestion of lactoperoxidase (SEQ ID No 2) is Shown in figure 5.

Peptide intensities were determined for the six unique peptides along with three other peptides displaying high peak area intensity. The data is shown below in Table 10.

Table 10: Summary of tryptic peptides from co-isolate lactoperoxidase.

n.d. not detected The most intense signature peptide ILGAFIQIITFR (SEQ ID No 9) and DGGIDPLVR (SEQ ID No 10) peptide were chosen for synthesis and initial assay validation. The DGGIDPLVR peptide appeared to give a higher and more consistent signal in the assay, however the amount calculated was lower than expected. For this reason, another signature peptide was chosen for synthesis. The GLQTVLK (SEQ ID No 11) peptide gave a good signal in co-isolate digests and appeared not to be affected by partial cleavage. Also, the Glutamine residue (Q) did not appear to be susceptible to deamination.

Lysosomal alpha-mannosidase:

In silico tryptic digestion of Lysosomal alpha-mannosidase (Q29451) using the Peptide Mass programme (https://web.expasy.org) identified 31 peptides, 7 to 16 amino acids in length. Elimination of peptides containing either cysteine or methionine residues reduced the total to 24 peptides. Nine of these peptides contained glutamine residues (Q) which are prone to deamination, leaving a total of fifteen potential diagnostic peptides as shown in Table 11. Table 11: Physical properties of Lysosomal alpha-mannosidase in-silico tryptic peptides

The online BLAST search in UniProt (www.uniprot.org) and NCBI (www.ncbi.nhn.nih.gov) were used for evaluating the specificity of the selected signature peptides. Peptides unique to Lysosomal alpha-mannosidase were chosen for further evaluation.

To determine which peptide was best identified by tryptic digestion and LC-MS/MS analysis, a sample of co-isolate was digested, and the raw data analysed using PEAKS Studio v8.5. The PEAKS Studio software gives an indication of the total peptide intensity providing information on the most abundant diagnostic peptides. Peptides showing a larger area intensity could mean that the peptide ionises and chromatograms better than other peptides, or that the sequence is more accessible/receptive to the enzyme providing better enzyme efficiency. In many ways, this is the best method for determining the best peptide for AQUA analysis.

The peptide coverage map for the tryptic digestion of Lysosomal alpha-mannosidase (SEQ ID No 3) is shown in Figure 6. Peptide intensities were determined for the ten unique peptides that showed good water solubility along with three other peptides displaying high peak area intensity. The data is shown below in Table 12.

The two most intense signature peptides WGPETLLLR (SEQ ID No 12) and (FQVIVYNPLGR (SEQ ID No 13) were chosen for synthesis and initial assay validation. The WGPETLLLR peptide appeared to give the higher and more consistent signal in the assay.

Table 12: Summary of tryptic peptides from co-isolate lysosomal alpha-mannosidase.

Immunoglobulin Gamma (subclass 1)

In-silico tryptic digestion of Immunoglobulin gammal heavy chain constant region (ABE68619) using the Peptide Mass programme (https://web.expasy.org) identified eight peptides, 6 to 16 amino acids in length. Elimination of peptides containing either cysteine or methionine residues reduced the total to seven potential diagnostic peptides as shown in Table 13.

Table 13: Physical properties of Immunoglobulin gammal heavy chain in-silico tryptic peptides

The online BLAST search in UniProt (www.uniprot.org) and NCBI (www.ncbi.nhn.nih.gov) were used for evaluating the specificity of the selected signature peptides. Peptides unique to Immunoglobulin gamma were chosen for further evaluation.

To determine which peptide was best identified by tryptic digestion and LC-MS/MS analysis, a sample of co-isolate was digested, and the raw data analysed using PEAKS Studio v8.5. The PEAKS Studio software gives an indication of the total peptide intensity providing information on the most abundant diagnostic peptides. Peptides showing a larger area intensity could mean that the peptide ionises and chromatograms better than other peptides, or that the sequence is more accessible/receptive to the enzyme providing better enzyme efficiency. In many ways, this is the best method for determining the best peptide for AQUA analysis.

The peptide coverage map for the tryptic digestion of Immunoglobulin gammal heavy chain (SEQ ID No 4) is shown in Figure 7.

Peptide intensities were determined for the seven unique peptides. The data is shown below in Table 14. Table 14: Summary of tryptic peptides from co-isolate lactoperoxidase.

n.d. not detected

Only four peptides were detected that were suitable for AQUA analysis. The two most intense signature peptides EPQVYVLAPPQEELSK (SEQ ID No 14) and VHN EGLPAPIVR (SEQ ID No 15) were chosen for synthesis and initial assay validation. The only concern about the

EPQVYVLAPPQEELSK peptides was that it demonstrated non-tryptic cleavage producing the peptide VLAPPQEELSK. This appeared to be ~30% of the total peptide. Unsurprisingly, the VHN EGLPAPIVR gave the higher and more consistent signal in the assay, but the amount calculated was lower than expected. For this reason, another peptide was chosen for synthesis and validation. The VVSALR (SEQ ID No 16) peptide gave a good signal in co-isolate digests and appeared to be not affected by partial cleavage. Angiogenin

In-silico tryptic digestion of angiogenin-1 (P10152) using the Peptide Mass programme (https://web.expasy.org) identified four peptides, 6 to 14 amino acids in length. Elimination of peptides containing either cysteine or methionine residues reduced the total to four potential diagnostic peptides as shown in Table 15.

Table 15 Physical properties of angiogenin in-silico tryptic peptides

The online BLAST search in UniProt (www.uniprot.org ) and NCBI (www.ncbi.nhn.nih.go v) were used for evaluating the specificity of the selected signature peptides. Peptides unique to angiogenin were chosen for further evaluation.

To determine which peptide was best identified by tryptic digestion and LC-MS/MS analysis, a sample of co-isolate was digested, and the raw data analysed using PEAKS Studio v8.5. The PEAKS Studio software gives an indication of the total peptide intensity providing information on the most abundant diagnostic peptides. Peptides showing a larger area intensity could mean that the peptide ionises and chromatograms better than other peptides, or that the sequence is more accessible/receptive to the enzyme providing better enzyme efficiency. In many ways, this is the best method for determining the best peptide for AQUA analysis.

The peptide coverage map for the tryptic digestion of angiogenin-1 (SEQ ID No 5) is shown in Figure 8. From this peptide map, it is evident that the cleavage pattern is not clean with multiple non-specific peptides being generated. This is not ideal. Some of the modifications are minor, and it is the relative intensity of the unmodified peptide compared to the various modified species, which is important. However, each modified species reduces the amount of quantitative peptide that can be detected. Peptide intensities were determined for the four peptides along with their relative percentage of total intensity. The data is shown below in Table 16.

Table 16: Summary of tryptic peptides from co-isolate angiogenin-1.

n.d. not detected

From this analysis, only two signature peptides were considered to fit the criteria for peptide selection:

YIHFLTQHYDAKPK (SEQ ID No 17)

NTFIHGNK (SEQ ID No 18)

Ribonuclease-4

In-silico tryptic digestion of ribonuclease-4 (P15467) using the Peptide Mass programme

(https://web.expasy.org) identified six peptides, 6 to 13 amino acids in length. Elimination of peptides containing either cysteine or methionine residues reduced the total to three potential diagnostic peptides as shown in Table 17. Table 17: Physical properties of angiogenin in-silico tryptic peptides

The online BLAST search in UniProt (www.uniprot.org) and NCBI (www.ncbi.nhn.nih.gov) were used for evaluating the specificity of the selected signature peptides. Peptides unique to RNase4 were chosen for further evaluation.

To determine which peptide was best identified by tryptic digestion and LC-MS/MS analysis, a sample of co-isolate was digested, and the raw data analysed using PEAKS Studio v8.5. The PEAKS Studio software gives an indication of the total peptide intensity providing information on the most abundant diagnostic peptides. Peptides showing a larger area intensity could mean that the peptide ionises and chromatograms better than other peptides, or that the sequence is more accessible/receptive to the enzyme providing better enzyme efficiency. In many ways, this is the best method for determining the best peptide for AQUA analysis.

The peptide coverage map for the tryptic digestion of RNase4 (SEQ ID No 6) is shown in Figure 9. Similar to the analysis for angiogenin, it can be seen from the peptide map of Figure 9 that the cleavage pattern is not clean with multiple non-specific peptides being generated. Peptide intensities were determined for the three peptides along with their relative percentage of total intensity. The data is shown below in Table 18. Table 18: Summary of tryptic peptides from co-isolate angiogenin-1.

n.d. not detected

Only one signature peptide fits the criteria for peptide selection:

FNTFIHEDLWNIR (SEQ ID No 19). EXAMPLE 3 - Determination of proteins in product by MRM analysis

Based on the identification of six target proteins in example 1 and the subsequent identification of the unique signature peptides of each target proteins (example 2), the following

methodology was developed to identify two or more of target proteins in a sample containing a milk protein by M RM analysis.

The following analytical standards were prepared/obtained for this analysis:

Peptide calibration standards from Biomatik CO As

• Lactoferrin calibration standard: LTF-WK (SVDGKEDLIWK, molecular mass 1289.43 Da).

Purity 96.72%. Peptide content 65.79%.

• Lactoperoxidase calibration standard : LPO-GK (GLQTVLK, molecular mass 757.95 Da).

Purity 98.50%. Peptide content 73.18%.

• Lysosomal alpha-mannosidase calibration standard : LAM-LR (WGPETLLLR, molecular mass 1084.27 Da). Purity 96.78%.

• Immunoglobulin G calibration standard : IgGl-VR (VVSALR, molecular mass 643.77 Da).

Purity 98.52%. Peptide content 72.91%.

• Angiogenin calibration standard : ANG-YK (YIHFLTQHYDAKPK, molecular mass 1761.20 Da). Purity 98.66%. Peptide content 71.74%.

• RNase4 calibration standard: RN4-FR (FNTFIFIEDLWN IR, molecular mass 1704.60 Da).

Purity 95.01%. Peptide content 74.77%.

Peptide isotope standards from Biomatik (COAs)

• Lactoferrin isotope standard : LTF-WKFI (S*VDGKEDLIWK, molecular mass 1295.59 Da).

Purity 96.12%.

• Lactoperoxidase isotope standard : LPO-GKFI (GLQI*VLK, molecular mass 763.91 Da).

Purity 95.46%.

• Lysosomal alpha-mannosidase isotope standard: LAM-LRFI (WGPET*LLLR, molecular mass 1091.46 Da). Purity 95.25%.

• Immunoglobulin G isotope standard: IgGl-VRFI (VVS*ALR, molecular mass 647.82 Da).

Purity 98.01%. • Angiogenin isotope standard : ANG-YKH (YIHF*LTQHYDAKPK, molecular mass 1767.40 Da). Purity 95.01%.

• RNase4 isotope standard: RN4-FRH (FNTFIHED*LWN IR, molecular mass 1711.80 Da).

Purity 98.15%.

Primary stock standards

The following method was followed in preparing the primary stock standard used in this analysis:

1. Make the peptide up in 50% of the required volume of 20% acetonitrile.

2. If the peptide solubilises make up the remaining volume with 20% acetonitrile.

3. If there is an issue with solubility check the pi of the peptide.

4. If the peptide is basic (i.e. pi =10.1), then add 1 mL of formic acid to acidify the solution and make up the remaining volume with 20% acetonitrile.

5. If the peptide is acidic (i.e. pi = 4.6), then add 5 mL of 1M NaOH to basify the solution and make up the remaining volume with 20% acetonitrile.

1 mg/mL Peptide calibration standards

Weigh out 1 ± 0.005 mg of each specific calibration peptide into a 1.7 mL Lo-Bind tube. Dilute to 1 mL with 20 % v/v acetonitrile following the instructions above. Store as 100 mL aliquots at -20 °C in a 0.5 mL Lo-Bind tube.

1 mg/mL Peptide internal standards (ISTD)

Weigh out 1 ± 0.005 mg of each specific ISTD peptide into a 1.7 mL Lo-Bind tube. Dilute to 1 mL with 20 % v/v acetonitrile following the instructions above. Store as 100 mL aliquots at -20 °C in a 0.5 mL Lo-Bind tube.

Note: 1 mg/mL ISTD is the equivalent of 1000 ppm ISTD.

Calculation of Peptide concentration

It is important to take into account both peptide purity and peptide content when calculating the peptide concentration from a measured weight of peptide powder.

LTF-WK Purity = 96.72% Peptide content = 65.79%

lmg/mL = 1000 x 0.9613 x 0.6579 = 636.32 mg/mL

100 mg/L = 100/0.63632 = 157.1 mL LPO-GK

Purity = 98.50% Peptide content = 73.18%

lmg/mL = 1000 x 0.9850 x 0.7318 = 720.82 mg/mL

100 mg/L = 100/0.72082 = 138.7 mL

LAM-LR

Purity = 96.78% Peptide content = unknown%

lmg/mL = 1000 x 0.9678 x 1 = 967.8 mg/mL

20 mg/L = 20/0.9678 = 20.7 mL

IgGl-VR

Purity = 98.52% Peptide content = 72.91%

lmg/mL = 1000 x 0.9852 x 0.7291 = 718.31 mg/mL

20 mg/L = 20/0.71831 = 27.8 mL

ANG-YK

Purity = 98.66% Peptide content = 71.74%

lmg/mL = 1000 x 0.9866 x 0.7174 = 707.78 mg/mL

100 mg/L = 100/0.70778 = 141.3 mL

RN4-FR

Purity = 95.01% Peptide content = 74.77%

lmg/mL = 1000 x 0.9501 x 0.7477 = 710.39 mg/mL

100 mg/L = 100/0.7104 = 140.8 mL

Secondary stock standards

Secondary stock standards were also prepared according to the following: 100 mg/L High Calibration Standard.

Add 157.1 mL 1 mg/mL LTF-WK (9.6.1)

Add 138.7 mI_ 1 mg/mL LPO-GK (9.6.2)

Add 141.3 mL 1 mg/mL ANG-YK (9.6.5)

Add 140.8 mL 1 mg/mL RN4-FR (9.6.6)

To 422.1 mL 20% acetonitrile.

2 mg/L High Calibration Standard .

To 20 mL 100 ppm High Calibration Standard add 980 mL 20% acetonitrile.

20 mg/L Low Calibration Standard.

Add 20.7 mί 1 mg/mL LAM-LR

Add 27.8 mL 1 mg/mL IgGl-VR

To 951.5 mL 20% acetonitrile.

2 mg/L Low Calibration Standard

To 100 mL 20 mg/L Low Calibration Standard add 900 mL 20% acetonitrile.

Isotope Calibration Standards

Isotope calibration stock standards were also prepared according to the following:

40 ppm Stock Isotope Standard

To 40 mL each of 1 mg/mL LTF-TKH, 1 mg/mL LPO-FRH, 1 mg/mL LAM-LRH, 1 mg/mL IgG-VRH,

1 mg/L ANG-YKH, and 1 mg/mL RN4-FRH add 720 mL 20% acetonitrile.

4 ppm Working Isotope standard

To 100 mL 40 ppm Stock Isotope Standard add 900 mL 20% acetonitrile.

Preparation of Calibration Standards

1 mL of fresh calibration standards were prepared into 1.5 mL Lo-bind tubes, capped and mixed and stored at -20°C. The standards can be used for up to one month as long as the same batch of 4 ppm internal standards are used to quantify the test samples. The ten-point standard curve is a dual standard curve consisting of both High and Low Calibration Standards. This enables a single calibration run to cover the proteins of interest, such as lactoferrin and lactoperoxidase proteins as well as report on the remaining less abundant proteins.

The volumes of each calibration standard component are tabulated below.

Table 7: Volumes of each calibration standard component

Preparation of test samples

The test samples were prepared according to the following method:

For the cationic protein base product, 20 mg ± 0.2 mg was accurately weighed into a 2 mL Lo- bind tube and made up to 20 mg/mL with 1 mL of GuHCI reducing buffer. The sample was mixed until all base powder had been solubilised .

For the detection of lactoferrin and lactoperoxidase dilute the sample 10-fold with GuHCI reducing buffer to 2 mg/mL (i.e. 100 mL of 10 mg/mL + 900 mL of GuHCL reducing buffer).

For Milk Protein Powder (MPP) 100 mg ± 0.2 mg was accurately weighed into a 2 mL Lo-bind tube and made up to 100 mg/mL with 1 mL type-1 water (10% w/v). The sample was mixed until all milk powder has been solubilised.

Digestion for LTF, LPO, ANG and RNase4 in the cationic protein base powder

The following methodology was followed for digestion of LTF, LPO, ANG and RNase4 in the cationic protein base powder:

Transfer 100 mL of diluted 2 mg/mL base powder solution into a 2 mL Lo-Bind tube.

Add 20 mL of 500 mM IAA solution. Mix and spin. Place in the dark for 30 min.

Add 720 mL of 50 mM ABC solution.

Add 50 mL of acetonitrile. Mix well

Add 50 mL of 4 ppm isotope internal standard (ISTD).

Add 20 mL of 1 mg/mL trypsin solution. Mix well and pulse spin.

Place on 37 °C shaking incubator overnight for 16 h. Set shaking speed to 400 rpm.

Spin and add another 20 mL of 1 mg/mL trypsin solution. Mix well and pulse spin.

Place in a 37 °C shaking incubator for a further 8 h. Set shaking speed to 400 rpm.

Spin samples and terminate the reaction with the addition of 20 mL of formic acid .

Digestion for Low abundance proteins in base powder

For detection of lower abundance proteins in the base powder, the following methodology was followed : Transfer 125 mL of 20 mg/mL base powder solution into a 2 mL Lo-Bind tube.

Add 20 mL of 500 mM IAA solution. Mix and spin. Place in the dark for 30 min.

Add 695 mL of 50 mM ABC solution.

Add 50 mL of acetonitrile. Mix well

Add 50 mL of 4 ppm isotope internal standard (ISTD).

Add 20 mL of 1 mg/mL trypsin solution. Mix well and pulse spin.

Place in a 37 °C shaking incubator overnight for 16 h. Set shaking speed to 400 rpm.

Pulse spin and add another 20 mL of 1 mg/mL trypsin solution. Mix well and pulse spin.

Place in a 37 °C shaking incubator for a further 8 h. Set shaking speed to 400 rpm.

Spin samples and terminate the reaction with the addition of 20 mL of formic acid.

Digestion in Milk Protein Powder

Transfer 200 mL of 100 mg/mL MPP solution into a 2 mL Lo-Bind tube.

Lyophilise sample in a rotary centrifugal speed-vac for 3 h.

Add 125 mL of GuHCI reducing buffer and place on 30 °C shaking incubator for a further 1 h to solubilise the sample. Set shaking speed to 600 rpm.

Check sample after 30 min. Vortex and spin to aid in solubilisation.

Add 20 mL of 500 mM IAA solution. Mix and spin. Place in the dark for 30 min.

Add 690 mL of 50 mM ABC solution.

Add 50 mL of acetonitrile. Mix well

Add 50 mL of 4 ppm isotope internal standard (ISTD).

Add 20 mL of 1 mg/mL trypsin solution. Mix well and pulse spin.

Place on 37 °C shaking incubator overnight for 16 h. Set shaking speed to 400 rpm.

Pulse spin and add another 20 mL of 1 mg/mL trypsin solution. Mix well and pulse spin.

Place in a 37 °C shaking incubator for a further 8 h. Set shaking speed to 400 rpm.

Spin samples and terminate the reaction with the addition of 20 mL of formic acid .

Sample Extraction

The samples were extracted by centrifuging digested samples at 12,000 x g for 5 minutes. Using a 200 mL Lo-Retention pipette tip, 200 mL aliquot of supernatant was transferred into glass insert for peptide detection by MS analysis.

Analytical Procedure

The following analytic procedure was followed to identify and detect the target proteins using tandem Mass spectrometry.

General Instrument Setup

TSQ Quantiva operation conditions

• Positive ion voltage: 3500 V

• Ion transfer tube temperature: 400 °C

• Vaporizer temperature: 350 °C

• CID Gas: 1.5 mTorr

• Q1 resolution (FWHM): 0.7

• Q3 resolution (FWHM): 0.7

Column

Hypersil Gold C18 column (100 x 2.1mm, 1.9 pm particle size; ThermoScientific) fitted with a Krudkatcher filter for 2.1 mm I.D.

Gradient Conditions

Mobile Phase A: 0.2 % formic acid in type-1 water

Mobile Phase B: 0.2 % formic acid in acetonitrile

Column temperature: 60 °C

Injection volume: 5 mL

Flow rate: 0.6 mL/min.

The gradient values are shown in Figure 3.

Table 8: Gradient conditions

MRM conditions for ion detection

The following conditions were set for analysis of target protein in a sample

Table 9: Conditions for analysis of target proteins

QT; quantifying ion, QF; qualifying ion.

Sequencing procedure for MS analysis

Preconditioning: A series of preconditioning injections in required to equilibrate/stabilize the mass spectrometry system before analysis. A sample bland and at least two test samples followed by the 2000/1000 mg/L calibration standard and two sample blank runs were run. The sequence structure consisted of the following:

• PreRun equilibration step

• Complete set of calibration standards (low to high).

• Blank run

• All samples (including QC samples).

• Blank run

• l x check standard (1000/500 pg/L) after every 10 injections of live samples

• Blank run

• Complete set of calibration standards (low to high).

• Shutdown sequence to leave the column in 60% acetonitrile for storage.

The results obtained from the run can then be used to identify the presence of and quantitate the amount of target proteins present in a sample.

Data Verification and Approval

Calibration Curve

Calibration curve fit (internal calibration): Quadratic with a 1/x weighing.

For a calibration curve to be acceptable the following criteria must be met:

Residuals of all included points must be within 10% of stated nominal value.

All calibration curves should have a minimum regression coefficient (R2) of >0.99.

A minimum of four points must be included in each calibration curve (not including the 0/0 pg/L calibration standard), and one must be at or below the required reporting limit.

Method-Specific Approval Criteria

There are a number of method specific criteria that must be met before the results can be validated and approved. Examples include:

If high blank levels are encountered, consider the possibility of carry over due to an instrument problem or a contamination issue during extraction. Carry over should be less than 1 %, determined by assessing the peak area of any analyte present in the first 0/0 pg/L calibration standard run directly after the highest calibration standard. Check that the levels of any residues in the replicate sample are within 10 % of their mean.

Acceptable check standard recoveries are between 80-110 %. If response drift greater than 20% is observed, notify a senior analyst immediately.

Internal standard area counts in normal samples should be no less than 60 % of calibration standards depending on the interaction with sample matrix and peptide solubility.

All detected residues must occur within the stated calibration range. If samples contain positive residues exceeding the highest calibration point, they must be diluted so they fall within the calibration range and re-analysed.

It will be appreciated that other method specific criteria can be included, and the above is not to be taken as an exhaustive list.

Data Calculation

Once the results have been obtained and approved, the quantification of the target proteins can be calculated. Raw data is reported in pg/L. Results are multiplied by the parent/peptide mass ratio for each protein to give the amount of the parent protein in pg/L.

The amount of the parent protein is then multiplied by the dilution factor to give a w/w value.

• For base powder LTF, LPO, ANG, and Rnas4 were diluted 5000-fold.

• For base powder LAM, and IgG were diluted 800-fold.

• For Milk Protein Powder the dilution was 50-fold.

EXAMPLE 4 - ADDITIONAL TESTING:

Additional testing of the product can be performed to determine any further

bioactive/therapeutic properties of the product and provided for consideration to an overall index rating for the product.

The methodology for each of the following test samples is described below: Enzyme activity test - Lactoperoxidase

Experiment protocol to determine of lactoperoxidase activity in powders, creams, lozenges and gums and test stability of lactoperoxidase in the product.

REAGENTS REQUIRED:

a. Assay Buffer - lOOmM potassium phosphate (pH 5.4)

b. Enzyme Diluent - 40mM potassium phosphate buffer, 0.25% (w/v) BSA, 0.5% (v/v) Triton X-100

c. 5.5mM ABTS substrate solution

d. 0.35% (w/w) hydrogen peroxide solution (H2O2)

e. 10 mg/ml lactoperoxidase enzyme solution or extract (positive control sample, optional)

Preparation of reagents:

a. Weigh 1.36 g of potassium dihydrogen phosphate (MWt 136.09) and dissolve in 100 ml of Milli-Q water, adjust to a pH of 5.4 with KOH (requires one or two drops of 1M KOH)

b. Transfer 40 ml of reagent 'a' to a beaker containing 0.25 g of bovine serum albumin and 500 mI of Triton X-100. Make up to 100 ml with Milli- Q water. Use as enzyme diluent for high dilutions of enzyme.

c. Weigh 30 mg of ABTS (Sigma Product No A-1888 -2, 2'-Azino-bis (3- Ethylbenzthiazoline-6-Sulfonic Acid), Diammonium Salt, or 3xl0mg tablets - Sigma Product A9941) and dissolve in 10 ml of Assay buffer, (i.e. 3mg/ml) PREPARE FRESH

d. Dilute approximately 130 mI of 27% H2O2 in 10 ml of Milli-Q water.

Confirm the concentration by diluting a sample 1/10 and measuring the absorbance at 240nm (quartz cuvette). The extinction coefficient is 0.043 pmol 1 ml cm at 240nm. (JBC 245, pp2409-13, 1970). For example, if the 1/10 dilution has an absorbance at 240nm = 0.45, the concentration is = 34 x 0.45/0.043 pg/ml = 355.8 pg H 2 0 2 /ml = 0.035%.) e. Weigh 30 mg of IDP and dissolve in 3 ml of reagent 'b'. As a guide using a 1/1000 dilution of the enzyme gives a suitable reaction rate in the test system.

Note: weights and volumes can be adjusted to suit logistics of a particular test run provided concentrations prescribed above are adhered to.

Preparation of samples:

a. IDP and co-isolate (freeze-dried powder)

Using a 4-place balance accurately weigh about 0.1 g of the dry powder and add Enzyme Diluent to a final weight of about 2.5g for a final concentration between 40 and 50 mg/ml. Make further 1/10,000 dilution in Enzyme Diluent to a final concentration of 40 to 50 micrograms/ml. Add 25 mI of this 1/10,000 dilution to the assay as below. b. Creams, Toothpastes and Gels

Accurately weigh about lg and suspend in 10ml of Enzyme Diluent (lOOmg/ml).

Vortex to ensure the sample is thoroughly resuspended.

When IDP has been added at 1% - 1.5% the sample should be diluted 1/400 and 25 mI added to the assay as below.

If the amount of lactoperoxidase is unknown the sample should be diluted 1/10, 1/100 and 1/1000 to find the appropriate amount that produces a reasonable rate of reaction. c. Gums and Lozenges

Accurately weigh 1 or 2 pieces and crush/macerate in 10ml of Enzyme Diluent. Dilute as appropriate for analysis dependent on the amount of IDP in each piece.

PROCEDURE:

Pipette the following reagents into a suitable cuvette:

Blank

Reagent b (Enzyme Diluent) 25 mI

Reagent c (ABTS Solution) 700 mI

Reagent d (H202) 25 mI

Equilibrate buffers to 25 ° C. Add first two reagents, mix well and monitor the absorbance at 405nm using a suitably thermostatted spectrophotometer. Add hydrogen peroxide (reagent d) to start the reaction and monitor the absorbance at 405nm for approximately 2 minutes.

NOTE: - the reaction rate will be affected by temperature, concentration of ABTS and concentration of peroxide, therefore these must all be controlled carefully for results to be compared from one day to another. Mixing in the cuvette is important to ensure homogeneity, especially when adding small volumes of reagent and repeat draw and dispense cycles with an autopipette can be used for mixing purposes.

Obtain the DA 405 nm/rninute using the maximum linear rate for Test and Blank.

CALCULATIONS:

Unit definition:

One unit will oxidize 1.0 mmole of 2, 2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) per minute at pH 5.4 at 20°C.

Equations:

0

d

3

0

FINAL ASSAY CONCENTRATIONS:

In a 0.75 ml reaction mix, the final concentrations are 5.1 mM 2, 2'-azino-bis (3- ethylbenzthiazoline-6-sulfonic acid) and 0.01% (w/w) hydrogen peroxide.

Antimicrobial activity test

Experiment protocol to determine the level of antimicrobial activity in bioactive protein samples in vitro. The test will allow comparison of the potency of antimicrobials present in samples containing bioactive milk proteins.

Culture maintenance:

Acquire cultures from the NZM R, ATCC, DSM or a culture collection. The strains used are Escherichia coli NZMR 916 (ATCC 259222 / DSM 1103), and Staphylococcus aureus NZM R 87 (ATCC 91442 / DSM 683, used in UMF testing of honey). At a minimum, replaced with stock from a culture collection every 2 years. Resuscitate cultures as per ESR's instructions. Grow overnight in TSB. Aliquot 500ul broth culture and 500mI 40% glycerol into several 2ml screw top tubes. Alternately use CryoBeads as per manufacturer's instructions. Store at -80. Every 3 months streak from a tubes to a TSA slope and plate. Discard the plate and re-streak from the slope on a weekly basis.

Procedure:

1. 5 hours prior to setting up assay inoculate 10ml TSB with E. co/i 916 and 10ml TSB with S. aureus 87. Incubate at 37°C.

2. Make NaSCN stock. Dissolve 0.8g of NaSCN in 10ml water.

3. Add 100mI of NaSCN stock solution to 9.9ml water (-1 dilution).

4. Dilute 1ml of the -1 dilution in 9ml water (-2 dilution, in reservoir).

5. Dissolve 200mg IDP in 10 ml TSB (11 am).

6. Heat at 40°C for 20 min with shaking (11.00 am). 7. Make dilution series as per table and add to plate, starting with the lowest concentration at the bottom of the plate (1.00pm). Make E. coli dilutions first, then dilute IDP 1:1 to make S. aureus dilutions.

8. Add 50mI of -2 dilution to each sample and control well.

9. Adjust E. coli and S. aureus cultures to OD600 0.15 to 0.25. Dilute cultures to -6.

Plate out IOOmI -6 dilution on TSB. Add 100mI of -2 dilution to 10ml TSB. Inoculate three replicates of dilution series with 10mI E. coli and three with S. aureus in each well. Add media only, IDP only and NaSCN only positive and negative controls.

10. Pop any bubbles with sterile tweezers. Seal plate with PlateSeal.

11. Incubate in plate reader at 37°C for 18 hours taking absorbance readings at 600nm every 20 minutes (plate reader Protocol AMP 600).

12. Determine CFU/well inoculum and, repeat any runs with an inoculum less than 250 or more than 500 cfu/well, or where more than 1 out of four of any given control fails.

13. Repeat three times on different days.

Record the results in the attached recording sheet.

Note: following addition of TSB or -2 dilution concentration is halved resulting in the final concentration.

Plate layout:

Record the contents of each well of each run in the following table.

Recording sheet:

The results obtained can then be recorded in the following recording sheet: Sample ID:

Run One

Date of setup: Batch TSB:

Gen5 experiment file:

Lowest concentration of inhibition of E. coli 916:

Lowest concentration of inhibition of S. aureus 87:

E. coli 916 OD: Inoculum: Score:

S. aureus 87 score: OD: Inoculum: Score:

Run Two

Date of setup: Batch TSB:

Gen5 experiment file:

Lowest concentration of inhibition of E. coli 916:

The entire disclosures of all applications, patents and publications cited above and below, if any, are herein incorporated by reference.

Reference to any prior art in this specification is not, and should not be taken as, an acknowledgement or any form of suggestion that that prior art forms part of the common general knowledge in the field of endeavour in any country in the world.

The invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, in any or all combinations of two or more of said parts, elements or features. Where in the foregoing description reference has been made to integers or components having known equivalents thereof, those integers are herein incorporated as if individually set forth.

It should be noted that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the spirit and scope of the invention and without diminishing its attendant advantages. It is therefore intended that such changes and modifications be included within the present invention.