Mass Spectrometry-

Field of the Invention

The present invention relates to sample plates for mass

spectrometry and their uses, and in particular to sample plates for use in surface assisted laser desorption ionization mass spectrometry .

Background of the Invention

Ease of-use, instrumental robustness, high sensitivity, high resolution and buffer tolerance have made matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-ToF MS a mainstream mass spectrometry tool that is particularly useful for the analysis of large molecules in proteomics, glycomics, polymer research and microbiology. Typically, soft ionization of analytes (without the excessive fragmentation characteristic to many other ionization techniques) is achieved by co-crystallising the analyte with an organic matrix (for example, 2 , 6-dihydroxy benzoic acid) that strongly absorbs at the laser excitation wavelength.

MALDI is typically a two-step process. Rapid heating by

nanosecond pulsed laser irradiation leads to matrix ablation and evaporation which carries the analytes into the gaseous phase. Analyte ion formation can follow several different pathways and often a distinction between matrix ion formation (primary path) and analyte ion formation in the plume (secondary path) can be made. ^1,2 Only a very small fraction of desorbed analytes are ionized and ionization yields (in positive mode) depend on various factors such as fluence, analyte basicity, temperature, matrix type etc.

While MALDI-ToF MS has proved invaluable for the analysis of large molecules and for proteomics, the analysis of small molecules for metabolomics , drug discovery and tissue imaging is hampered by the spectral interference of matrix-derived species of masses below 700Da. In addition, the requirements of MALDI- ToF MS for co-crystallization and solubility of analyte and matrix have motivated the search for alternatives based on soft laser desorption ionization. -

In 1999 Suizdak and co-workers reported efficient desorption- ionization of analytes from a nanostructured porous silicon, made from a silicon wafer by electrochemical etching. Analyte molecules are efficiently trapped into the surface pores and easily volatized under laser irradiation due to the high

absorption coefficient of nanoporous silicon and the limited heat dissipation of the thin walled cavities which leads to a rapid and very localized heating at low laser fluence.

In surface assisted laser desorption ionization mass spectrometry (SALDI-MS) , instead of an organic matrix, nanostructured surfaces with specific physical properties are chosen that enhance desorption and ionization of deposited analytes. Absorption at the laser excitation wavelength is a requirement but sub-micron patterning of the material, together with increases the surface area thereby concentrating the analyte, is advantageous. A number of nanoparticles and surface materials have been

explored ^33,1,4 and suggested for SALDI-MS including, semiconductor metal and metal oxide ^0- ' nanoparticles, nanostructured gold ^8-12 or platinum ¹³ surfaces and nanoparticles ¹⁴ , germanium oxide surfaces, etched porous silicon ^1,15,1,16 , silicon nanowires ¹⁷ and -posts ¹⁸ , the carbon allotropes ¹⁹ fullerene, graphite, carbon nanotubes or nanodiamonds , nanostructured gold surfaces. These surfaces have been employed for the qualitative analysis of carbohydrates ³ ^' ²⁰ , steroids ²⁰ , lipids ²¹ " ²² , peptides ^23,24 and for tissue imaging. ²⁵

While many of these surfaces have proved useful in SALDI MS techniques, they often require very precise manufacturing conditions, for example, clean rooms and carefully controlled atmospheric conditions^ and/or are expensive to produce,

sometimes because of the materials used or the manufacturing processes themselves. As a result, use of these nanostructured surfaces as sample plates for mass spectrometry may be

constrained by cost restrictions, or the lack of facilities or relevant expertise. Other disadvantages of some of these known materials include a lack of stability under storage at ambient conditions, low analytical reproducibility, insufficient

sensitivity, interference through background ions and/or low dynamic range.

WO 2004/113924 relates to a method of capturing peptide from gels onto surfaces formed from polycarbonate or polyester that can then be studied using mass spectroscopy.

WO 2006/083151 relates to sample plates formed from etched and coated substrates. Stainless steel is the only form of steel mentioned.

WO 2007/133724 describes chips for use in mass spectroscopy with self-assembled monolayers and capture agents for binding to analyte. The substrate on which the layers are assembled can be metallic; the only metals specified are gold and silver.

US 2004/024545) relates to LDI sample plates coated with a thin layer of carbon. The underlying substrate can be metallic, but only conventional stainless steel and gold coated steel plates are described.

US 2004/0038423 relates to coated stainless steel MALDI plates.

GB 2378755 relates to a support for holding conventional

stainless steel MALDI plates.

GB 2381068 discloses laser etched and coated MALDI plates relating to conventional stainless steel as the substrate.

Accordingly, there remains a need in the art for sample plates capable of use for the sensitive analysis of a wide range of analytes by techniques such as surface enhanced laser desorption mass spectrometry. Summary of the Invention

Broadly, the present invention is based on the finding that sample plates for surface enhanced laser desorption ionization (LDI) mass spectrometry having sample surfaces formed from weathering steel, and in particular plates of cut and polished weathering steel sheets, can be employed as sample plates for the sensitive analysis of a wide range of analytes by surface enhanced laser desorption mass spectrometry. Weathering steels, commonly referred to by the trade name COR-TEN®, are low-alloy steels that, due to doping with nickel, chromium, phosphorous, silicon and calcium, are protected against corrosion through the formation of a nanostructured iron oxide and/or hydroxide mesh or other nanostructure such as a plurality of iron oxide and/or iron hydroxide nanowires, nanotubes, nanorods and/or nanofibers.

Other elements that may be used in the formation of these weathering steels include copper, manganese, vanadium,

molybdenum, sulphur and rare earth elements. Weathering steel is commercially available, of comparatively low cost and, for the present uses and methods, shows good stability with respect to storage, and additionally stability under storage at ambient conditions, low cost of production (including the possibility of having single use slides), and low levels of background ions.

Accordingly, in a first aspect, the present invention provides a sample plate for surface enhanced laser desorption ionization (LDI) mass spectroscopy which comprises a sample surface formed from a weathering steel.

The sample plates of the present invention may be used in different forms of laser desorption ionization (LDI) mass spectrometry, including surface assisted laser desorption ionization (SALDI) mass spectrometry or surface enhanced laser desorption ionization (SELDI) mass spectrometry.

Conveniently, the sample plate is cut from a sheet of weathering steel. In some aspects, the sample surface of the slide has a passivated layer as obtainable by removing an outer iron oxide layer on the sample surface of the weathering steel and then exposing the resultant surface to humid atmospheric conditions to promote slow formation of rust crystals on the surface. This enables sample plates to be produced easily and at low production costs .

Additionally or alternatively, the sample plates enable laser desorption ionization (LDI) mass spectrometry to be carried out which are capable of detecting analyte species present in nanomole quantities, and more preferably detecting analyte species present in picomole quantities, and even more preferably detecting analyte species present in femtomole quantities.

In a further aspect, the present invention provides a method of making a sample plate for laser desorption ionization (LDI) mass spectrometry, the method comprising:

cutting a sample plate from a sheet of weathering steel; removing an outer oxide layer from the sample surface of the sample plate; and

passivating the sample surface of the sample plate to produce an LDI-active surface.

Optionally further derivatisation of the sample plate can be performed.

In a further aspect, the present invention provides a sample plate obtainable by a methods described herein.

In a further aspect, the present invention provides the use of a sample plate of the present invention in the detection of an analyte using laser desorption ionization (LDI) mass

spectrometry .

In a further aspect, the present invention provides a method of detecting an analyte using laser desorption ionization (LDI) mass spectrometry, the method comprising: depositing the analyte onto the sample surface of a sample plate of the present invention;

irradiating the sample surface with a laser to desorb and ionize the analyte;

detecting the ionized analyte.

An advantage of the present invention is that while the sample plates are sufficiently inexpensive to be single use plates, they are also sufficiently easy to re-use for example by washing the sample surface, e.g. with eOH, hexanes, and/or water.

Possible applications for sample plates made of weathering steel can be expected in small molecule analysis in drug discovery and academic research, tissue imaging by MS, as single use sample plates in kits for the quantification of metabolites, food ingredients (example lactose) or the glycoanalysis of

glycoproteins by isotopic dilution.

Embodiments of the present invention will now be described by way of example and not limitation with reference to the accompanying figures. However various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

"and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example "A and/or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

Unless context dictates otherwise, the descriptions and

definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described. Brief Description of the Figures

Figure 1. SALDI- S of synthetic bi-antennary glycan equipped with aminopentyl linker 1. Figure 2. A Comparison of different surfaces in LDI-MS detection of biantennary glycan 1. B Limit of detection for glycan standard X on weathering steel.

Figure 3. Background ions on weathering steel.

Figure 4. SALDI-MS on small molecules, metabolites and drug like compounds at detected at picomolar quantities.

Figure 5. Lamivudine analysis on weathering steel.

Figure 6. Top: profile of serum metabolites in positive ion mode (top) .

Figure 7. Direct lactose detection in milk.

Figure 8. Direct lactose quantification in diluted crude milk sample by isotopic dilution.

Figure 9. SALDI-MS analyses comparing the lipid profiles of human breast and bovine milk. The bottom spectrum shows a

magnification of the most abundant triglycerides.

Figure 10. Glycan analysis on a hydrophobic weathering steel plate .

Figure 11. TEM images at various magnifications showing the effect of slides prepared using treatment Tl, stored in the lab at ambient conditions. Figure 12. SEM images of COR-TEN steel after storage at room temperature for 48 h and then after subsequent 1 h immersion in deionised water at various magnifications. Figure 13. XPS analysis of slides shown in Figure 14, Upper image: polished slide stored at room temperature and XPS analysis of COR-TEN steel after storage at room temperature. Lower image: image and XPS spectra of slide stored at room temperature and subsequent immersion in deionized water for 1 h

Figure 14. SEM images of COR-TEN steel after immersion in a NaCl 24% w/v solution in deionized water for Ih and dried prior to analysis.

Figure 15. SEM images of COR-TEN steel after immersion in a H ₂ S0 ₄ 2% w/v solution in deionized water for lh and dried prior to analysis .

Figure 16. Characterization of a polished weathering steel plate, before (a) and after (b) coating with OTS.

Figure 17. Matrix-free LDI mass spectra collected from mouse brain tissue sections on a polished weathering steel slide. The tissue sections had not been processed after mounted in the target.

Figure 18. Matrix-free LDI mass spectra collected from mouse brain tissue sections on a OTS coated weathering steel slide. The tissue sections had not been processed after mounted in the target .

Figure 19. Matrix-free LDI mass spectra collected after an aqueous washing of the brain tissue sections on a OTS coated weathering steel slide. The slide was dried with a stream of Ar after washing .

Figure 20. LDI-MS spectra of IgG glycans (up), and IgG glycans in the presence of a labeled glycan standard (down) using a OTS- coated slide. Figure 21. LDI-MS spectra of the mixture of 8 glycan standards using a OTS-coated slide.

Figure 22. A mixture of 8 glycan standards analyzed by LDI-MS in weathering steel slides after polishing and washing (middle) , after TMO coating (up) and by MALDI-MS using DHB matrix (down) .

Figure 23. LDI-MS spectra of the 8 glycan standards in TMO-coated slides without using mass deflection, 0-2200 Da.

Detailed Description

Weathering Steels

The term weathering steel is understood in the art and describes a group of steel alloys that, owing to their alloy content, exhibit increased resistance to atmospheric corrosion through the formation of a corrosion resistant oxide patina. Weathering steels are also often referred to as atmospheric corrosion resistant steels, self-protecting steels, self-passivating steels, and weather resistant steels.

Weathering steels are low alloy steels. Commonly used alloying elements include nickel, copper, chromium, phosphorous, silicon and calcium, although other alloying elements, including

manganese, vanadium, molybdenum, sulphur and rare earth elements may also be used. Preferred alloying combinations may include Cu-Cr and Cu-Cr-P- (Ni ) .

Accordingly, in some embodiments of the present invention, the weathering steel is a weathering steel comprising one or more of the following alloying elements in the composition percentage range given: carbon (0 - 0.2%), silicon (0 - 1%), manganese

(0 - 2%), phosphorus (0 - 2%), sulphur (0 - 0.05%), chromium (0 - 2%), copper (0 - 1%), nickel (0 - 1%), vanadium (0 - 2%) and aluminium (0 - 0.1%). In some embodiments of the present

invention, the weathering steel is a weathering steel comprising one or more of the following alloying elements in the composition percentage range given in parentheses: carbon (0.12 - 0.19%), silicon (0.25 - 0.75%), manganese (0 - 1.5%), phosphorus (0 -

1.5%), sulphur (0 - 0.04%), chromium (0.3 - 1.25%), copper (0.25

- 55%), nickel (0 - 0.65%), vanadium (0 - 1.2%) and aluminium (0.02 - 0.06%) . In some embodiments of the present invention, the weathering steel is a weathering steel comprising one or more of the following alloying elements in the composition percentage range given in parentheses: carbon (0.12 - 0.19%), silicon (0.25

- 0.75%), manganese (0 - 1.5%), phosphorus (0 - 1.5%), sulphur (0 - 0.04%), chromium (0.3 - 1.25%), copper (0.25 - 55%), nickel (0 - 0.65%), vanadium (0 - 1.2%) and aluminium (0.02 - 0.06%) .

In some embodiments, the weathering steel must comprise at least one of the following alloying elements in the given range: Al total >0.020%, Nb = 0.015 - 0.060%, V = 0.02 - 0.12%, Ti = 0.02 - 0.10%.

In some embodiments, the weathering steel comprises at least 0.2%, preferably at least 0.3%, chromium, and in some preferred embodiments the percentage chromium content is in the range 0.3 - 1.5%, preferably 0.3 - 1.25%. In some embodiments, the weathering steel comprises at least 0.1%, preferably at least 0.2%, copper, and in some preferred embodiment the percentage copper content is in the percentage range 0.1 - 0.6%, preferably 0.25 - 0.55%. In some embodiments, the weathering steel

comprises at least 0.0.1%, preferably at least 0.02%.

Examples of particular weathering steels suitable for use according to the present invention include, but are not limited to, COR-TEN A® and COR-TEN B@; EN 10155 grade weathering steels available from COR-TEN®, and other COR-TEN® weathering steels,

09CuP, 09CuPCrNi-A, and 09CuPCrNi-B (commercially available from YUSHENG IRON AND STEEL CO., LIMITED), and ferrite-perlite and ferrite-bannite weathering steels.

Weathering steels undergo a self-passivation process. The present inventors have found that the surface formed in this passivation process desorbs and ionizes adsorbed analytes under irradiation with a laser at 355nm very efficiently without the addition of any organic matrix compound. No use for matrix-free desorption ionization employing this material as a sample plate has been reported before. Studies have shown that the rust on some weathering steels can be divided into two different layers, an outer layer with a- and γ-FeOOH phases and an protective inner layer with enriched Cu and P content, which is and a nanometric mesh of FeOOH. Synchrotron radiation X-ray analysis has revealed that this protective inner layer is primarily composed of nanometric Cr-goethite crystals "containing surface-adsorbed and/or inter- granular CrO _x ^3~2x complex anions" ²⁸ within Fe(0,OH) ₆ network leads to a distortion of the rust structure. ²⁹ The heterogeneously-inserted surface chromium leads to the formation of very fine crystals rust with a consistent size distribution due to the increase in nucleation sites and a decrease of the critical radius of nucleation r*. ²⁹ Without wishing to be bound by any particular theory, the inventors believe that it may be this defined surface nanostructure formed on the low alloy steel as a protective layer that is responsible for effective laser energy absorption and energy transfer to analytes deposited onto the surface, resulting in the utility of weathered steel sheets for use in SALDI-MS methods described herein.

The sample plates of the present invention may be used for laser desorption ionization (LDI) mass spectrometry and are capable of detecting molecular species with mass/charge (m/z) ratios below 700, more preferably below 600, more preferably below 500, more preferably below 400, with signal to noise ratios greater than 5

More preferably, the sample plates of the present invention may be used for laser desorption ionization (LDI) mass spectrometry is capable of detecting molecular species with mass/charge (m/z) ratios below 700, more preferably below 600, more preferably below 500, more preferably below 400, with signal to noise ratios greater than 25.

More preferably, the sample plates of the present invention may be used for laser desorption ionization (LDI) mass spectrometry is capable of detecting molecular species with mass/charge (m/z) ratios below 700, more preferably below 600, more preferably below 500, more preferably below 400, with signal to noise ratios greater than 50.

More preferably, the sample plates of the present invention may be used for laser desorption ionization (LDI) mass spectrometry is capable of detecting molecular species with mass/charge (m/z) ratios below 700, more preferably below 600, more preferably below 500, more preferably below 400, with signal to noise ratios greater than 100.

More preferably, the sample plates of the present invention may be used for laser desorption ionization (LDI) mass spectrometry is capable of detecting molecular species with mass/charge (m/z) ratios below 700, more preferably below 600, more preferably below 500, more preferably below 400, with signal to noise ratios greater than 100. More preferably, the sample plates of the present invention may be used for laser desorption ionization (LDI) mass spectrometry is capable of detecting molecular species with mass/charge (m/z) ratios below 700, more preferably below 600, more preferably below 500, more preferably below 400, with signal to noise ratios greater than 200.

Preparation of Sample Plates

Sample plates suitable for use in methods of the present

invention may be prepared according to the following general method. A sample plate of suitable size, for example, 2.75 x 7.5 cm and 1 mm thickness, is laser cut from a sheet of weathering steel, for example COR-TEN A© or COR-TEN B®. The outer oxide layer of the sample surface is then removed from the sample plate using abrasion, for example using a suitable rotating metal brush or sandpaper. Passivation of ^' the surface to form the fine nanostructured iron oxide/hydroxide surface advantageous for use in the methods of the present invention may then occur either through storage at, for example, room temperature, or be

accelerated through immersion in water for, for example, about 1 h. Typically, the sample surface may be immersed in water for between 30 minutes and 24 hours, and more preferably for between 30 minutes and 2 hours. This may be followed by a drying step, conveniently at 18-25 °C. Exposing the sample surface to humid atmospheric conditions to promote slow formation of rust crystals on the surface. By way of example, the passivating step may last between one week and one year. If desired, the surface may then be derivatised, for example, through the application of a hydrophobic layer such as a layer of octadecylsilane or

trimethoxy (octadecyl ) silane.

The surface of the plate may be derivatised, for example, through the application of a tetraethylorthosilicate film. The film can then be further derivatized for example by immersion in a silane to promote silanization, the silane is preferably an alkyl silane for example, but not limited to, methyl silane. Silanization can be affected in a solvent for example an organic solvent,

preferably dichloromethane . Silanization can be performed for example at room temperature. Silanization can be performed for example from 1 to 48 hours, preferably about 24 hours. After the silanization the resulting slide can be rinsed for example with an organic solvent, preferably dichloromethane. The slide may then be dried prior to loading the analyte for example under an argon flow. It may be preferable to store the slide in a desiccator until used.

Derivatisation to produce a microarray

Microarrays are now a commonly used format for most high- throughput screening applications in genomics, proteomics and glycomics and the ability to analyse them by more than a single readout method is desirable to broaden their applications.

US 14/203611, which is herein incorporated by reference in its entirety, describes novel microarrays and methods of making these novel microarrays which may be assembled on a sample plate according to the present invention to provide a microarray suitable for use in SALDI-MS methods as described herein.

Accordingly, in a further aspect, the present invention provides a method of making a microarray on a surface of a sample plate according to the present invention, the method comprising:

(a) providing sample according to the present invention;

(b) forming a support layer of hydrophobic molecules attached to the surface of the sample plate;

(c) forming a layer of linker molecules on the surface of the sample plate, wherein the linker molecules comprise a hydrophobic group capable of non-covalently binding to the support layer and a reactive functional group; and

(d) printing a plurality of binding agents at a plurality of locations on the sample plate, wherein the binding agents comprise a functional group capable of reacting in situ on the microarray with the reactive functional group of the linker molecules to covalently link the binding agents to the linker molecules immobilized on the sample plate, thereby forming the microarray.

The present invention further provides a microarray supported on a sample plate according to the present invention as obtainable by this method or any other method described in US 14/203611.

Use of the sample plates in kits with isotopically tagged glycan standards

There exists an unmet need for improved methods for rapidly and easily analysing the content of released glycan mixtures and solutions. The identification and quantification of glycans and particular glycan signatures may be useful for the diagnosis of numerous diseases and disorders.

PCT/EP2014/056737 , which is herein incorporated by reference in its entirety, describes methods and materials for the

identification and quantification of the glycan content of sample using isotopically-labelled glycan standards (tagged standards), and provides methods for the preparation of suitable isotopologues for use in these methods. In particular,

PCT/EP201 /056737 provides a kit for identifying a glycan in a sample, the kit comprising:

(a) a tagged standard, the tagged standard comprising one or more isotopically-labelled glycans;

(b) instructions for doping a sample suspected of

containing a glycan with the tagged standard to obtain a doped sample and analysing the doped sample using mass spectrometry. Methods of synthesis and preparation of suitable isotopically- labelled glycan standards are provided more fully in

PCT/EP2014/056737.

In a further aspect, the present invention provides a kit as described in PCT/EP2014/056737 further comprising a sample plate formed of weathering steel as described herein according to any described embodiment of the present invention. In some preferred embodiments, the sample plate comprises a hydrophobic layer, for example, an organic silane, e.g. an alkyl silane such as

octadecylsilane (OTS) or an alkoxy silane, such as

trimethoxy (octadecyl) silane (TMO) , to facilitate analysis of the glycan mixtures. It is envisaged that such a kit, comprising one or more tagged standards and a sample plate formed of weathering steel, may have utility as a convenient and disposable kit for the rapid analysis of samples suspected of containing glycans.

Producing sample slides from a weathering steel

Initially, slides cut from weathering steel sheets of 2.5 mm thickness and immersed in deionized water for 1 h and showing visible signs of corrosion resulted in a remarkable S/N ratio of up to 700 for picomolar detection of the synthetic N-glycan 1 deposited as a standard in the comparison of potentially LDI- active surfaces (Figure lb) . The surface fine structure before and after water treatment was analysed and the formation of a mesh of nanosized wires of approximately 1-2 micrometer length observed by scanning electron microscopy (SEM)for the water treated sample see Figure 12, which shows the steel surface before and after treatment with dionized water for 1 h.

The passivation process may be accelerated by immersing the slides in deionised water, tap water and artificial sea water, wiping them clean with a lint free cloth.

A larger batch of samples plates (100 slides) of 2.5x7.5 cm size and 1 mm thickness were laser cut from Corten® steel sheets and the outer iron oxide layer removed by oil-free polishing with a rotating metal brush.

The slides were kept in a humidity chamber during months prior to use in LDI experiments to promote slow passivation of the exposed metal surfaces but without visible rust formation. The

performance of these surfaces in the matrix free LDI- S detection of a glycan 1 was compared with a standard stainless steel ALDI- plate, a stainless steel sample plated scratched with sand paper (grain size 600) and a commercial LDI plate based on silicon nanowire structures (NALDI) .

Figure 2A shows that the weathering steel sample plate exhibited the highest S/N ratio of all surfaces tested with a nearly 4.5 fold gain in sensitivity over the standard sample plates at considerably lower fluence. Figure 2B shows that the even at femtomole quantities, the same standard can be detected with good signal to noise ratio on the weathering steel sample plate that had been immersed in water for 1 hour and dried before analysis.

The sensitivity of the weathering steel plates according to the present invention compares extremely favourably with scratched and polished stainless steel plates, as can be seen in Figure 2A. This sensitivity allows analytes present in only very small quantities to be detected, as is demonstrated in Figure 2B.

Figure 3 shows a remarkably clean background spectra in the absence of analyte with major background ions sodium (m/z =23) and potassium (m/z= 39), which can be used as internals standards for mass calibration. The plate for this experiment plate had not been immersed in water prior to analysis, and was made from polished plain weathering steel.

Surface assisted laser desorption ionisation mass spectrometry experiments

The inventors then evaluated the scope of this new surface for the matrix-free surface assisted laser desorption ionisation mass spectrometry in the analysis of a wide variety of analytes including carbohydrates, lipids, drugs and metabolites in pure form or within a more complex matrix. Slides were divided into 64 wells by a silicon mask, which simplified analyte deposition and spot identification in the mass spectrometer via a virtual sample plate specified to the plate coordinates.

Figure 4 shows mass spectra of example compounds after deposition of 1 μΐ of a 4mM aqueous solution which amounts to picomole quantities of analytes onto the weathering steel sample plates. The selection included the drugs lamivudine (m/z= 229.1), a reverse-transcriptase inhibitor used for the treatment of

Hepatitis B and HIV and , the metabolites lysine (m/z = 146.1), choline (m/z= 104.1), creatinine (m/z= 113.1), hypotaurine (m/z= 109.0) and creatine (m/z = 131.0), and small molecules as lactose (m/z = 342.1) and hexaethyleneglycol (m/z = 282.2) . Most compounds were detected as sodium and potassium adducts; only choline was detected as the quaternary ammonium ion and for creatinine additionally the protonated species was observed.

Potassium (m/z= 39) was found in most spectra as a prominent background peak.

For lamivudine analysis, serial dilution was performed to study the sensitivity of the platform. Down to 50 picomole of analyte deposited onto the weathering steel plate by SALDI-TOF MS of could be detected routinely with excellent S/N ratios of 231 ([M+Na] ⁺ ) and 718 ( [M+K] ⁺ =353.27 , respectively. Analysis of serum metabolites

As an example for the analysis of a more complex matrix, serum sample was diluted with MeOH to precipitate present proteins and then an aliquot of the supernatant spotted directly onto the weathering steel plate and analysed. SALDI-ToF analysis in both negative (not shown) and positive mode (Figure 6) showed a large number of ions corresponding to major serum metabolites without any interference of background ions. Major prominent LDI-spectra ions in the LDI spectra were putatively assigned after a search in the open-access metabolite mass spectral database Metlin (http://metlin.scripps.edu/) to the high-concentration serum metabolites glutamine ( [M+H] ⁺ =146.99) , glucose ( [M+Na] ⁺ =203.05, ( [M+K] ⁺ =219.03) , oleic acid ( [M+Na] ⁺ =305.08, ( [M+K] ⁺ =321.05 ) , palmitoyl glycerol ( [M+Na] ⁺ =353.27) , cholesterol ([M+Na]"

=409.35) , diacylglyerides DG 16:0/16:0 ([M+Na] ⁺ = 619.53 and DG 18:0/18:0 { [M+Na] ⁺ =647.56 anf triacylglycerides TG 18:1/16:1/16:1 ( [M+Na] ⁺ =853.73 and TG 18:1/18:1/16:1 ( [M+Na] ⁺ =881.76. Lactose analysis in milk

As an example of a rapid and quantitative analysis of a low- weight analyte within a complex matrix lactose was chosen.

Lactose is an important constituent of cows milk and the

widespread inherited intolerance towards lactose among consumers has prompted the dairy industry to offer low-lactose and lactose free milk and derived dairy products. Quick and inexpensive methods for lactose quantification are therefore needed in the production process of lactose free products and in general for post production food analysis. Analysis of a diluted milk samples by SALDI-MS on weathering steel produced a clean spectra for lactose and galactose and glucose (same mass) in the case of lactase treated lactose-free milk (Figure 7) . By combining SALDI-MS on weathering steel sample plates with an internal isotopically tagged lactose standard, the inventors have

developed an isotopic dilution method for the absolute

quantification of lactose in milk samples. Dilution of crude samples of lactose free and normal low-fat milk and addition of all- ¹³ C labelled lactose standard allows the quantification of lactose for both sodium and potassium adducts even in lactose- free samples containing less than 10 mg lactose per 100 mL of milk, as is shown in Figure 8.

Figure 9 shows a comparison of the lipid profile of a human breast milk sample (Sigma Aldrich) compared to a sample of bovine milk. The magnification shows a shift of to higher masses for the most abundant triglycerides in human milk sample compared to bovine milk (Figure 9, bottom spectra), with TG ( 16 : 0/18 : 1/18 : 1 ) being the most abundant.

N-glycans are an important post-translational modification of therapeutic antibodies and proteins, which can strongly influence Fc-receptor binding in antibody-dependant cell mediated

cytotoxicity (ADCC) , protein immunogenicity, protein stability and protein circulatory halflife and clearance. Rapid,

inexpensive and quantitative methods for the analysis of protein glycosylation are needed not only for the batch to batch quality control in the highly regulated process of glycoprotein

development and production and but also in the increasingly importantly field of glycan biomarker discovery and detection. SALDI-MS in combination with isotopically tagged glycans could provide the quantitative mass spectrometric methods required for the rapid, and inexpensive glycoanalysis of iriAbs and other therapeutic glycoproteins with minimal sample preparation.

Complex synthetic glycans were ionized efficiently on weathering steel sample plates even without any derivatisation . N-glycans released by treatment with peptide N-glycosidase F from a human serum IgG sample were analyzed on a weathering steel plate which had been coated with octadecylsilane (OTS) to form a hydrophobic layer. ³⁰ As seen in Figure 10 a complete profile of complex N- glycans present on serum IgGs could be acquired on a sample containing less than 100 picomole glycans. LDI-MS glycan analysis using different hydrophobic coatings on weathering steel slides.

OTS coated slides for LDI-MS glycan analysis .

A sample of glycans released from human IgG by PNGaseF treatment was loaded directly in the OTS-coated slide (200 pmol of total glycan content) and analyzed by LDI-MS. In a second experiment, the glycan sample was spiked with an amount of labeled glycan standard G2F. The mixture of natural glycans and the standard was loaded in the slide and analyzed by LDI-MS (Figure 20) .

Also, a mixture of 8 labeled N-glycan standards was prepared in _d H ₂ 0 as a 50 μΜ solution of each glycan. The mixture was loaded in the OTS-coated slide (0.5 μΕ, 25 pmol of each glycan) and analyzed by LDI-MS (Figure 21) . Polished slides and TMO (trimethoxy octadecyl silane) coated slides for LDI-MS glycan analysis .

Polished slides washing: Polished Corten slides were thoroughly cleaned by immersion and sonication in hexane and isopropanol (10 minutes each) avoiding any paper rubbing. After this procedure, the clean slides were dried under an argon flow and stored in a desiccator until used.

TEOS (Tetraethyl orthosilicate) primer coating protocol: ^31,32 The slides previously cleaned and dried were coated with a tetraethylorthosilicate film which facilitates the further silanization process. The coating process is performed by immersion of the substrates in an ethanolic

tetraethylorthosilicate solution (1:1) containing 1 % of acetic acid for 30 seconds followed by one minute in water. After this time the slides are dried under an argon flow. The TEOS - water cycle is then repeated 3 times until the desired film properties are obtained. Finally, the coated substrates are cured for 1 hour at 80 °C and then stored in a desiccator until used.

TMO (Trimethoxy (octadecyl) silane) silanization conditions: The TEOS-coated slides were silanized by immersion in a 30 mM silane solution in dry dicholoromethane at room temperature for 24 hours. After this time, the slides were thoroughly rinsed with dichloromethane, dried under an argon flow and stored in a desiccator until used.

Detection of a mixture of 8 glycan standards using a TMO-coated weathering steel slide is shown in Figure 22. The signal to noise ratio is comparable to that of the polished weathering steel slides. Both the TMO-coated and polished weathering steel slides exhibit a similar signal to noise ratio than that observed using standard MALDI techniques with a DHB matrix.

Figure 23 demonstrates that TMO-coated weathering steel slides can be used to detect 8 glycan standards in high resolution without using mass deflection. The ability to analyse samples without mass deflection enables the detection of compounds present in only small amounts or compounds that readily decompose upon ionisation. Further, non-targeted mass spectrometry studies such as tissue imaging, metabolomics or where little background ions are expected are preferably carried out without mass deflection, for example by employing SALDI-MS.

Figures 11, 12, 14 and 15 show SEM images at varying

magnifications following a variety of passivation processes.

These processes include passivation occurring through immersion for one hour in water, artificial sea water or acid followed by 48 hours at room temperature. Figure 11 shows XPS analyses of a COR-TEN A® surface after passivation at room temperature and of a COR-TEN A® surface which has undergone accelerated passivation through immersion in water for one hour followed by storage at room temperature for 48 hours. The spectra shows the elemental composition at different sputtering times which relates directly to surface depth. Upper XPS spectra shows very low iron oxide level confined to the outer layer while the lower spectra of the oxidised surface shows a thick iron oxide layer protruding far into the material. Figure 16 shows spectra of glycan 1 on a weathering steel plate passivated through immersion in water for one hour followed by storage at room temperature for 48 hours. Ions for iron oxide background peaks can be observed when deflector is turned off.

Polished and OTS coated weathering steel slides surface

characteriza tion

The inventors then evaluated the difference in surface

characteristics of polished vs coated weathering steel slides using OTS-coating as an example.

Polished and OTS-coated slides (0.8 um roughness) . The surface topology of both surfaces was analysed by scanning electron microscopy (SE ) while depth dependant changes in the elemental surface composition due to corrosion were studied by X-ray photoelectron spectroscopy (XPS) .

SEM analysis of the polished untreated weathering steel sample showed an amorphous and scaly fine structure of the surface at the resolution studied. The depth-dependant XPS analysis showed the presence of a thin iron oxide layer that was rapidly removed after only a few minutes of Argon sputtering, revealing the elemental iron layer.

Surface functionalisation with OTS resulted in slides with increased hydrophobicity as evident by contact angle

measurements, polished (θ= 65 ± 1°) and OTS-coated weathering steel slides (θ= 128 ± 1°) .

SEM analysis of the OTS-coated slide showed a change in the fine structure towards features with lower grain size. The inventors consider, not wishing to be bound by theory, that this may be a result of the controlled corrosion involving chloride ions of the OTS reagent (Figure 16b) . The stronger corrosion was confirmed by XPS analysis which showed iron only in the form of Fe ₂ 0 ₃ . Even after prolonged Argon sputtering only traces of elemental iron were revealed. Polished and OTS coated weathering steel slides: Application: LDI-MS imaging of brain tissues

The inventors then evaluated the difference in LDI-MS imaging observed with the polished vs OTS-coated weathering steel slides. A new batch of 1 mm thickness weathering steel slides was polished with a rotating metal brush obtaining a mirror-like surface (0.07 μπι surface roughness) . A number of the polished slides were coated with octadecylsilane (OTS) to form a

hydrophobic layer.

Plain weathering steel slides and OTS-treated hydrophobic 1mm thick slides were tested for LDI imaging of mouse brain sections. A single mouse brain was cut by a cryostat microtome into 4 urn and 20 μπι thick sections and thaw mounted onto weathering steel slides. The brain sections were fixed to the slides and were dried after removal from the freezer and stored under vacuum. The 4 μιτι sections were used for direct LDI of the dry tissue (Figure 17, polished slides; Figure 18, OTS-coated slides) .

Both slides provide mass data over a wide range of masses. The hydrophobic slides provide particular clear LDI images. Without wishing to be bound by theory, it is thought that the increased quality of the images relates to improved sensitivity which may also affect the S/N ratio. A possible explanation of this is that the UV-Vis absorbance of the hydrophobic slides is higher than the polished slides (100% vs 70% at 355 nm) .

Subsequently, the 20μπι sections mounted on hydrophobic weathering steel were removed from the slide using water washings and the slide analysed by LDI for the detection of the immobilized metabolites (stamp procedure, Figure 19) .

As can be seen from figure 19 that a range of metabolites were immobilized on the hydrophobic surface. The resulting low sample concentration could be analysed using the present method due to the sensitivity observed when employing weathering steel sample surfaces in mass spectroscopy.

Summary and Uses

Weathering steel, showing nanostructures formed in the

passivation process, can be used as a low-cost and reusable sample plate with good to excellent sensitivity for the detection of a broad range of analytes by surface assisted and matrix-free laser desorption ionisation mass spectrometry. Covalent

functionalisation of the surface using silane chemistry (e.g. OTS) employing standard protocols can easily change the

hydrophobicity or affinity of the surface towards certain analytes. Fabrication of sample plates with specifications required for use in MS analysis (flatness, purity) is estimated to be far lower than any of the LDI surfaces which have been or are currently commercialised and which require the use of clean room facilities for carrying out sophisticated multi-step surface modifications. Possible applications for sample plates made of weathering steel can be expected in small molecule analysis in drug discovery and academic research, tissue imaging by MS, as single use sample plates in kits for the quantification of metabolites, food ingredients (example lactose) or the

glycoanalysis of glycoproteins by isotopic dilution. Further covalent and non-covalent functionalisation of sample plates, e.g. with affinity probes can provide a surface for the

fabrication of (micro) arrays for matrix-free mass spectrometry based high throughput assays.

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