This application claims priority from and the benefit of United Kingdom patent application No. 1708835.2 filed on 2 June 2017. The entire content of this applications is incorporated herein by reference. FIELD OF THE INVENTION

The present invention relates generally to methods and systems of analysis, particularly to methods of imaging a sample. BACKGROUND

In mass spectrometry imaging, the spatial distribution of the composition of sample is visualised by analysing ions produced from multiple spatially separated regions of the sample. Mass spectrometry imaging is an established analytical tool for bimolecular research, enabling the accurate localisation of molecules directly from a tissue section. Mass spectrometry imaging using conventional techniques can be very time consuming. For example, the analysis of a sample deposited on a typical glass slide can take many hours and even days.

Lately, developments in mass spectrometry imaging have been accomplished using ambient ionisation techniques, such as desorption electrospray ionisation mass

spectrometry ("DESI-MS") and its derivatives. For example, DESI-MS has been used for imaging small molecules such as lipids and metabolites from a tissue section. In this case, the lipids and metabolites are primarily ionised to form singly charged ions. Because these molecules are relatively soluble, and do not substantially adhere to the section substrate, the secondary droplets containing the analyte material readily desorb or vaporise from the substrate. The DESI-MS technique is described for instance in R. Crooks et al. "Mass Spectrometry Sampling Under Ambient Conditions with Desorption Electrospray

Ionisation", Science, 2004, 306, 471-473. Some related techniques derived from DESI-MS are described in a survey article "Ambient Mass Spectrometry", Science, 2006, 31 1 , 1566- 1570. DESI-MS is also described in various patents and patent publications including US 7,847,244 (PURDUE RESEARCH FOUNDATION), US 8,203, 117 (PROSOLIA, INC.) and US 7,335,897 (PURDUE RESEARCH FOUNDATION).

However, mass spectrometry imaging data is (irrespective of the ionisation technique) very complex and its analysis is challenging. Furthermore, direct analysis techniques such as DESI-MS often have relatively low sensitivity due to the inherent large amounts of background chemical noise resulting from the solvent, etc. Because of these inherent sensitivity issues, it has not generally been thought possible or practical to apply direct imaging techniques such as DESI-MS to image larger and more complex

biomolecules such as proteins and peptides directly from tissue sections. Thus, larger biomolecules including proteins and peptides have conventionally been imaged using matrix-assisted laser desorption/ionisation ("MALDI") techniques. For example, MALDI analysis of proteins from tissue has been implemented to attempt to elucidate high mass to charge molecular distributions in tissues that may be related to tissue type and/or a disease state. However, MALDI techniques may require vacuum conditions for efficient ionisation. Furthermore, MALDI analysis techniques require a relatively time consuming matrix deposition sample preparation step. This step can give rise to significant variability in the experiment because the matrix can vary across the sample and between samples, and may be unstable over the timescale of an experiment. Moreover, existing analyses of such compounds may generally be preceded by various off line sample preparation or separation steps which again can be relatively time-consuming and may lead to significant variability in the experiments.

SUMMARY

According to a first aspect, there is provided a method of analysing peptides and/or proteins directly from a surface of a tissue sample comprising:

directing a spray of solvent droplets from a sprayer device onto a surface of the tissue sample, wherein the solvent droplets generate peptide and/or protein ions from the surface of the tissue sample by a process of desorption;

collecting at least a portion of the generating ions using a sampling orifice, wherein the sampling office is connected to an ion analyser; and

analysing the ions with the ion analyser.

In embodiments, one or more operating conditions of the sprayer device may be selected and/or controlled so as to promote ionisation by a process of desorption over ionisation via charge transfer. That is, the operating conditions may be selected and/or controlled so as to promote electrospray-type ionisation rather than sonic spray-type ionisation.

The generated ions may thus comprise multiply charged peptide and/or protein ions. For instance, at least some of the generated ions may have charge states greater than 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, or 24.

The tissue sample may comprise a tissue section such as a human or animal tissue section. The tissue sample (e.g. tissue section) may thus be analysed in order to determine a tissue type and/or a disease state of the tissue.

The solvent droplets may be charged. For instance, a voltage may be applied to the sprayer device in order to charge the solvent or the solvent droplets. For example, the sprayer device may comprise a spray capillary, as in a conventional electrospray ionisation ("ESI") type source, and a voltage greater than about 4 kV may be applied to the spray capillary in order to charge the solvent droplets. In embodiments, voltages between about 4 and 5 kV, such as voltages of about 4.5 kV, may be applied to the spray capillary. Advantageously, the tissue sample (e.g. tissue section) may be analysed directly without prior application of a matrix. Advantageously, the tissue sample may be analysed directly without any off line separation such as centrifugation.

The tissue sample (e.g. tissue section) may be analysed directly without enzymatic digestion. Thus, the ions may correspond to native intact peptides and/or proteins.

However, in embodiments, an enzyme such as protease may be applied to the tissue section prior to analysis to generate peptides, and wherein the peptides are analysed directly. The enzyme may be applied to the tissue section in situ. Thus, the method may comprise a prior step (i.e. before the step of directing the spray of solvents onto the surface of the tissue sample) of applying an enzyme such as protease to the surface of the tissue sample (e.g. tissue section) to generate peptides. The resulting peptides may then be analysed directly from the surface of the tissue sample (e.g. tissue section).

The tissue sample (e.g. tissue section) may be analysed under ambient or atmospheric conditions.

Liquid solvent may be provided to the sprayer device at a solvent flow rate of greater than about 1 μΙ_/ηιίη. The solvent flow rate may be within a range of between about: (i) 1 to 4 μΙ_/ηιίη; (ii) 2 to 4 μΙ_/ηιίη; or (iii) 2 to 3 μΙ_/ηιίη.

The solvent may comprise an organic solvent such as acetonitrile. Other suitable and electrospray compatible solvents may include dichloromethane (optionally mixed with methanol), dichloroethane, tetrahydrofuran, ethanol, propanol, methanol, nitromethane, toluene (optionally mixed with methanol or acetonitrile), or water. The solvent may further comprise an acid such as formic or acetic acid. For example, the solvent may comprise between about 0.2 and 0.4% by volume acid. Where the solvent comprises acetonitrile, the solvent may comprise a ratio by volume of acetonitrile:water of between about 50:50 and 90: 10, such as between about 60:40 and 90: 10, such as between about 70:30 and 90: 10, such as about 80:20.

The solvent may further comprise one or more additives for enhancing the generation of multiply charged species. For example, the solvent may comprise as additives DMSO or 3-NBA. Other suitable additives may include volatile salts or buffers such as ammonium acetate or ammonium bicarbonate. Various other additives including dimethylformamide (DMF), trifluoroacetic acid, heptafluorobutyric acid, sodium dodecyl sulphate, ethylenediaminetetraacetic acid, and involate salts or buffers such as sodium chloride and sodium phosphates may also be added.

The spray of solvent droplets may be generated using a nebulizing gas provided to the sprayer device, wherein the nebulizing gas is provided at a pressure within a range of between about 3 to 7 bar; suitably between about 5 to 7 bar; or more suitably between about 5 to 6 bar.

In embodiments, the sampling orifice may be heated. For instance, the sampling orifice may be heated at or to a temperature of above about 200 ⁰ C, such as a

temperature of above about: (i) 250 ⁰ C; (ii) 300 ⁰ C; (iii) 350 ⁰ C; or (iv) 400 ⁰ C. The sampling orifice may be heated at or to a temperature between about 300 and 1000 ⁰ C, such as between about 300 and 750 ⁰ C or between about 300 and 500 ⁰ C. More suitably, the sampling capillary may be heated at or to a temperature between about 400 and 1000 ⁰ C, such as between about 400 and 750 ⁰ C, between about 400 and 500 ⁰ C or between about 400 and 450 ⁰ C.

The ion analyser may typically comprise a mass spectrometer.

The mass spectrometer may comprise one or more ion guides.

The mass spectrometer may comprise one or more ion mobility separation devices and/or one or more Field Asymmetric Ion Mobility Mass spectrometer devices. For instance, the mass spectrometer may comprise an ion mobility separator provided upstream of a mass analyser. The method may thus comprise selecting one or more regions of ion mobility and mass or mass to charge ratio space, and displaying only mass spectra corresponding to the selected one or more regions. In this way, the analysis may be (effectively) limited to ions of interest. The method may also comprise only analysis species within the selected one or more regions. For example, an ion filter such as a mass filter may be used to restrict the ions that are mass analysed to those falling within the selected one or more regions.

The mass spectrometer may comprise one or more ion traps or one or more ion trapping regions.

The mass spectrometer may comprise a mass filter. For example, the mass spectrometer may comprise one or more mass filters selected from the group consisting of: (i) a multipole mass filter, such as a quadrupole mass filter; (ii) a 2D or linear quadrupole ion trap; (iii) a Paul or 3D quadrupole ion trap; (iv) a Penning ion trap; (v) an ion trap; (vi) a magnetic sector mass filter; (vii) a Time of Flight mass filter; and (viii) a Wien filter.

The mass filter may be provided downstream of the ion mobility device, and the method may comprise using the mass filter to select multiply charged ions for mass analysis. For instance, the method may comprise selecting ions having charge states of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, or 24. In

embodiments, the method may comprise selecting only such multiply charged ions for mass analysis.

The mass spectrometer may comprise a fragmentation or reaction device. For instance, the mass spectrometer may comprise one or more collision, fragmentation or reaction cells selected from the group consisting of: (i) a Collisional Induced Dissociation ("CID") fragmentation device; (ii) a Surface Induced Dissociation ("SID") fragmentation device; (iii) an Electron Transfer Dissociation ("ETD") fragmentation device; (iv) an Electron Capture Dissociation ("ECD") fragmentation device; (v) an Electron Collision or Impact Dissociation fragmentation device; (vi) a Photo Induced Dissociation ("PID") fragmentation device; (vii) a Laser Induced Dissociation fragmentation device; (viii) an infrared radiation induced dissociation device; (ix) an ultraviolet radiation induced dissociation device; (x) a nozzle-skimmer interface fragmentation device; (xi) an in-source fragmentation device; (xii) an in-source Collision Induced Dissociation fragmentation device; (xiii) a thermal or temperature source fragmentation device; (xiv) an electric field induced fragmentation device; (xv) a magnetic field induced fragmentation device; (xvi) an enzyme digestion or enzyme degradation fragmentation device; (xvii) an ion-ion reaction fragmentation device; (xviii) an ion-molecule reaction fragmentation device; (xix) an ion-atom reaction

fragmentation device; (xx) an ion-metastable ion reaction fragmentation device; (xxi) an ion-metastable molecule reaction fragmentation device; (xxii) an ion-metastable atom reaction fragmentation device; (xxiii) an ion-ion reaction device for reacting ions to form adduct or product ions; (xxiv) an ion-molecule reaction device for reacting ions to form adduct or product ions; (xxv) an ion-atom reaction device for reacting ions to form adduct or product ions; (xxvi) an ion-metastable ion reaction device for reacting ions to form adduct or product ions; (xxvii) an ion-metastable molecule reaction device for reacting ions to form adduct or product ions; (xxviii) an ion-metastable atom reaction device for reacting ions to form adduct or product ions; and (xxix) an Electron lonisation Dissociation ("EID") fragmentation device.

The method may comprise recording one or more parent ion mass spectra and one or more fragment or product ion spectra at each probed location of the surface of the tissue sample (e.g. tissue section).

The mass spectrometer may comprise a mass analyser selected from the group consisting of: (i) a quadrupole mass analyser; (ii) a 2D or linear quadrupole mass analyser; (iii) a Paul or 3D quadrupole mass analyser; (iv) a Penning trap mass analyser; (v) an ion trap mass analyser; (vi) a magnetic sector mass analyser; (vii) Ion Cyclotron Resonance ("ICR") mass analyser; (viii) a Fourier Transform Ion Cyclotron Resonance ("FTICR") mass analyser; (ix) an electrostatic mass analyser arranged to generate an electrostatic field having a quadro-logarithmic potential distribution; (x) a Fourier Transform electrostatic mass analyser; (xi) a Fourier Transform mass analyser; (xii) a Time of Flight mass analyser; (xiii) an orthogonal acceleration Time of Flight mass analyser; and (xiv) a linear acceleration Time of Flight mass analyser.

The mass spectrometer may comprise one or more energy analysers or electrostatic energy analysers.

The mass spectrometer may comprise one or more ion detectors.

The mass spectrometer may comprise a device or ion gate for pulsing ions; and/or a device for converting a substantially continuous ion beam into a pulsed ion beam.

The mass spectrometer may comprise a C-trap and a mass analyser comprising an outer barrel-like electrode and a coaxial inner spindle-like electrode that form an electrostatic field with a quadro-logarithmic potential distribution, wherein in a first mode of operation ions are transmitted to the C-trap and are then injected into the mass analyser and wherein in a second mode of operation ions are transmitted to the C-trap and then to a collision cell or Electron Transfer Dissociation device wherein at least some ions are fragmented into fragment ions, and wherein the fragment ions are then transmitted to the C-trap before being injected into the mass analyser.

The mass spectrometer may comprise a stacked ring ion guide comprising a plurality of electrodes each having an aperture through which ions are transmitted in use and wherein the spacing of the electrodes increases along the length of the ion path, and wherein the apertures in the electrodes in an upstream section of the ion guide have a first diameter and wherein the apertures in the electrodes in a downstream section of the ion guide have a second diameter which is smaller than the first diameter, and wherein opposite phases of an AC or RF voltage are applied, in use, to successive electrodes.

The mass spectrometer may comprise a device arranged and adapted to supply an AC or RF voltage to the electrodes. The AC or RF voltage optionally has an amplitude selected from the group consisting of: (i) about < 50 V peak to peak; (ii) about 50-100 V peak to peak; (iii) about 100-150 V peak to peak; (iv) about 150-200 V peak to peak; (v) about 200-250 V peak to peak; (vi) about 250-300 V peak to peak; (vii) about 300-350 V peak to peak; (viii) about 350-400 V peak to peak; (ix) about 400-450 V peak to peak; (x) about 450-500 V peak to peak; and (xi) > about 500 V peak to peak.

The AC or RF voltage may have a frequency selected from the group consisting of:

(i) < about 100 kHz; (ii) about 100-200 kHz; (iii) about 200-300 kHz; (iv) about 300-400 kHz; (v) about 400-500 kHz; (vi) about 0.5-1.0 MHz; (vii) about 1.0-1.5 MHz; (viii) about 1.5-2.0 MHz; (ix) about 2.0-2.5 MHz; (x) about 2.5-3.0 MHz; (xi) about 3.0-3.5 MHz; (xii) about 3.5-4.0 MHz; (xiii) about 4.0-4.5 MHz; (xiv) about 4.5-5.0 MHz; (xv) about 5.0-5.5 MHz; (xvi) about 5.5-6.0 MHz; (xvii) about 6.0-6.5 MHz; (xviii) about 6.5-7.0 MHz; (xix) about 7.0-7.5 MHz; (xx) about 7.5-8.0 MHz; (xxi) about 8.0-8.5 MHz; (xxii) about 8.5-9.0 MHz; (xxiii) about 9.0-9.5 MHz; (xxiv) about 9.5-10.0 MHz; and (xxv) > about 10.0 MHz.

The ion guide may be maintained at a pressure selected from the group consisting of: (i) < about 0.0001 mbar; (ii) about 0.0001-0.001 mbar; (iii) about 0.001-0.01 mbar; (iv) about 0.01-0.1 mbar; (v) about 0.1-1 mbar; (vi) about 1-10 mbar; (vii) about 10-100 mbar; (viii) about 100-1000 mbar; and (ix) > about 1000 mbar.

Analyte ions may be subjected to Electron Transfer Dissociation ("ETD") fragmentation in an Electron Transfer Dissociation fragmentation device. Analyte ions may be caused to interact with ETD reagent ions within an ion guide or fragmentation device.

Optionally, in order to effect Electron Transfer Dissociation either: (a) analyte ions are fragmented or are induced to dissociate and form product or fragment ions upon interacting with reagent ions; and/or (b) electrons are transferred from one or more reagent anions or negatively charged ions to one or more multiply charged analyte cations or positively charged ions whereupon at least some of the multiply charged analyte cations or positively charged ions are induced to dissociate and form product or fragment ions; and/or (c) analyte ions are fragmented or are induced to dissociate and form product or fragment ions upon interacting with neutral reagent gas molecules or atoms or a non-ionic reagent gas; and/or (d) electrons are transferred from one or more neutral, non-ionic or uncharged basic gases or vapours to one or more multiply charged analyte cations or positively charged ions whereupon at least some of the multiply charged analyte cations or positively charged ions are induced to dissociate and form product or fragment ions; and/or (e) electrons are transferred from one or more neutral, non-ionic or uncharged superbase reagent gases or vapours to one or more multiply charged analyte cations or positively charged ions whereupon at least some of the multiply charge analyte cations or positively charged ions are induced to dissociate and form product or fragment ions; and/or (f) electrons are transferred from one or more neutral, non-ionic or uncharged alkali metal gases or vapours to one or more multiply charged analyte cations or positively charged ions whereupon at least some of the multiply charged analyte cations or positively charged ions are induced to dissociate and form product or fragment ions; and/or (g) electrons are transferred from one or more neutral, non-ionic or uncharged gases, vapours or atoms to one or more multiply charged analyte cations or positively charged ions whereupon at least some of the multiply charged analyte cations or positively charged ions are induced to dissociate and form product or fragment ions, wherein the one or more neutral, non-ionic or uncharged gases, vapours or atoms are selected from the group consisting of: (i) sodium vapour or atoms; (ii) lithium vapour or atoms; (iii) potassium vapour or atoms; (iv) rubidium vapour or atoms; (v) caesium vapour or atoms; (vi) francium vapour or atoms; (vii) C ₆ o vapour or atoms; and (viii) magnesium vapour or atoms.

The multiply charged analyte cations or positively charged ions may comprise peptides, polypeptides, proteins or biomolecules.

Optionally, in order to effect Electron Transfer Dissociation: (a) the reagent anions or negatively charged ions are derived from a polyaromatic hydrocarbon or a substituted polyaromatic hydrocarbon; and/or (b) the reagent anions or negatively charged ions are derived from the group consisting of: (i) anthracene; (ii) 9, 10 diphenyl-anthracene; (iii) naphthalene; (iv) fluorine; (v) phenanthrene; (vi) pyrene; (vii) fluoranthene; (viii) chrysene; (ix) triphenylene; (x) perylene; (xi) acridine; (xii) 2,2' dipyridyl; (xiii) 2,2' biquinoline; (xiv) 9- anthracenecarbonitrile; (xv) dibenzothiophene; (xvi) 1 , 10'-phenanthroline; (xvii) 9' anthracenecarbonitrile; and (xviii) anthraquinone; and/or (c) the reagent ions or negatively charged ions comprise azobenzene anions or azobenzene radical anions.

The process of Electron Transfer Dissociation fragmentation may comprise interacting analyte ions with reagent ions, wherein the reagent ions comprise

dicyanobenzene, 4-nitrotoluene or azulene.

The mass spectrometer may be operated in various modes of operation including a mass spectrometry ("MS") mode of operation; a tandem mass spectrometry ("MS/MS") mode of operation; a mode of operation in which parent or precursor ions are alternatively fragmented or reacted so as to produce fragment or product ions, and not fragmented or reacted or fragmented or reacted to a lesser degree; a Multiple Reaction Monitoring ("MRM") mode of operation; a Data Dependent Analysis ("DDA") mode of operation; a

Data Independent Analysis ("DIA") mode of operation a Quantification mode of operation or an Ion Mobility Spectrometry ("IMS") mode of operation.

The electrodes may comprise electrodes which are formed on a printed circuit board, printed wiring board or an etched wiring board. For example, according to various embodiments the electrodes may comprise a plurality of traces applied or laminated onto a non-conductive substrate. The electrodes may be provided as a plurality of copper or metallic electrodes arranged on a substrate. The electrodes may be screen printed, photoengraved, etched or milled onto a printed circuit board or equivalent. According to an embodiment the electrodes may comprise electrodes arranged on a paper substrate impregnated with phenolic resin or a plurality of electrodes arranged on a fibreglass mat impregnated within an epoxy resin. More generally, the electrodes may comprise one or more electrodes arranged on a non-conducting substrate, an insulating substrate or a plastic substrate. According to embodiments the plurality of electrodes may be arranged on a substrate.

A plurality of insulator layers may be interspersed or interleaved between an array of electrodes. The plurality of electrodes may be arranged on or deposited on one or more insulator layers.

The method may further comprise a prior step of washing the sample using ethanol and/or chloroform. For example, the method may comprise a prior step of washing the sample using ethanol, followed by a step of washing the sample using chloroform.

The method may be a method of desorption electrospray ionisation mass spectrometry ("DESI-MS") imaging.

From another aspect, there is provided a method of imaging peptides and/or proteins directly from a tissue sample (e.g. tissue section) comprising a method

substantially as described herein, the method further comprising surveying the tissue sample so as to visualise the spatial distribution of peptides and/or proteins over the surface of the tissue sample. That is, the method may comprise probing or analysing a plurality of different positions or areas of the surface of the tissue sample in order to build up an image of the surface of the tissue sample.

From a further aspect, there is provided a system for performing a method substantially as described herein, the system comprising:

a sprayer device for generating a pneumatic spray of solvent droplets, wherein the spray of solvent droplets is directed, in use, onto a tissue sample (e.g. tissue section); and a sampling orifice connected to an ion analyser for receiving ions generated from the tissue sample by the pneumatic spray of solvent droplets.

The system may comprise a platform for receiving a tissue sample (e.g. tissue section). The platform may be moveable and/or the sprayer device may be moveable relative to the platform in order to allow analysis or visualisation of the entire surface of the tissue sample.

The sampling orifice may be heated. For example, the sampling orifice may be heated at or to a temperature of above about 200 ⁰ C, such as a temperature of above about: (i) 250 ⁰ C; (ii) 300 ⁰ C; (iii) 350 ⁰ C; or (iv) 400 ⁰ C. The sampling orifice may be heated at or to a temperature between about 300 and 1000 ⁰ C, such as between about 300 and 750 ⁰ C or between about 300 and 500 ⁰ C. More suitably, the sampling capillary may be heated at or to a temperature between about 400 and 1000 ⁰ C, such as between about 400 and 750 ⁰ C, between about 400 and 500 ⁰ C or between about 400 and 450 ⁰ C. One or more heaters or heating elements may be provided for heating the sampling orifice.

The sprayer device may comprise an electrospray ionisation source such as a desorption electrospray ionisation ("DESI") source.

The system may further comprise a control system for controlling an adjustable supply of a nebulizing gas and/or a liquid solvent to the sprayer device. In embodiments, the control system may control the pressure at which the nebulizing gas is provided to the sprayer device and/or the flow rate at which the solvent is provided to the sprayer device. Particularly, the nebulizing gas pressure and/or the solvent flow rate may be controlled within the ranges described above.

The ion analyser may comprise or be a mass spectrometer substantially as described above. That is, the mass spectrometer may comprise any (or all) of the features described above in relation to the first aspect. For example, the mass spectrometer may comprise an ion mobility separator. Furthermore, the mass spectrometer may comprise a mass filter and/or an ion fragmentation or reaction device substantially of any of the types described above.

The system may be arranged and adapted to perform any of the method steps described above and/or may comprise means suitable for performing any such method steps.

BRIEF DESCRIPTION OF THE DRAWINGS Various embodiments will now be described, by way of example only, and with reference to the accompanying drawings in which:

Fig. 1 schematically shows an example of a desorption electrospray ionization mass spectrometry ("DESI-MS") interface;

Fig. 2A shows the effects of ion mobility separation on the mass spectral peaks of interest, and Fig. 2B shows a plot of ion mobility (drift time) against mass to charge ratio obtained from a section of rat liver, with a region of certain high mobility species of interest derived from the tissue highlighted;

Fig. 3 shows the effects on various mass spectral peaks of interest of heating the sampling capillary;

Figs. 4A and 4B show the effects on various mass spectral peaks of interest of varying the nebulizer gas pressure;

Figs. 5A and 5B show the effects on various mass spectral peaks of interest of varying the solvent flow rate;

Figs. 6A and 6B show the effects on various mass spectral peaks of interest of varying the solvent composition, wherein the solvent comprises acetonitrile;

Figs. 7 A and 7B show the effects on various spectral peaks of interest of varying the solvent composition, wherein the solvent comprises methanol;

Figs. 8A and 8B compare the effects of acetonitrile and methanol as solvents; Figs. 9A and 9B illustrate the effects of adding 3-NBA to the solvent, and Figs. 10A and 10B illustrate the effects of adding DMSO to the solvent;

Figs. 1 1 A and 11 B show the effects on various spectral peaks of interest of varying the voltage applied to the spray capillary;

Figs. 12A and 12B illustrate the effect of washing the sample;

Figs. 13A and 13B show experimental results from a DESI-I MS-MS analysis of a full tissue section of mouse brain;

Figs. 14A and 14B show experimental results from a DESI-IMS-MS analysis of fresh frozen mouse liver tissue section; and Fig. 15 shows experimental results from a DESI-I MS-MS analysis of fresh frozen mouse brain tissue section wherein a solution of trypsin was applied on site to the tissue.

DETAILED DESCRIPTION

Various embodiments as will be described in more detail below are concerned with a method of desorption electrospray ionization mass spectrometry ("DESI-MS") analysis. According to DESI-MS techniques, analyte material is liberated from the surface of a sample at ambient pressure by directing a spray of charged solvent droplets 13 onto the sample. A typical DESI-MS interface for analysing a sample 20 is illustrated in Fig. 1. As shown, the interface generally includes a DESI sprayer 10 for directing the spray of charged solvent droplets 13 onto the sample 20 so as to cause molecules on the surface of the sample 20 to desorb and/or directly ionise and a sampling orifice such as a sampling capillary 30 arranged above the sample 20 in order to receive the ions that are generated from the sample 20 and pass these ions towards an ion analyser such as a mass spectrometer (not shown) for analysis. The sampling capillary 30 may be arranged to inhale or draw in the generated ions to improve the sampling efficiency.

It will be appreciated that various mass spectrometers may suitably be used for analysing the ions generated by the DESI process. In some embodiments, the mass spectrometer may comprise an ion mobility separation device for separating the ions according to their ion mobility prior to their mass analysis. The additional ion mobility separation may thus act to further resolve or separate the generated ions from each other thus increasing the analytical capacity of the ion analyser. It will be appreciated that this may be particularly important for complex proteomic analyses. Furthermore, the ion mobility separation may provide additional information about the analyte, as the ion mobility data may be complementary to the mass analysis. The combined use of ion mobility and mass analysis also allows for various ion manipulation and/or post-processing techniques that may advantageously simplify the analysis and/or provide further information. For example, the mass spectrometer may further comprise one or more mass filtering devices and/or ion fragmentation or reaction devices. The mass filtering devices may be operated in synchronism with the ion mobility separation device (where one is provided) in order to select certain mass ranges of interest e.g. corresponding to certain charge states of interest. The ion fragmentation or reaction devices may be used to fragment or react the parent analyte ions in order to generate a series of characteristic product or daughter ions which may be used to help identify or qualify the parent analyte ions.

As shown, a DESI sprayer 10 generally comprises a spray capillary that is provided with a pressurised supply of nebulizing gas 12 (e.g. nitrogen) and a flow of liquid solvent 1 1 for generating the spray 13 of droplets. Thus, in order to generate the solvent spray 13, a liquid solvent 11 is fed into the spray capillary alongside a high velocity nebulizing gas flow 12 so that the nebulizing gas acts to nebulize the solvent exiting the spray capillary. A voltage may be applied to the DESI sprayer 10, or to the flow of liquid solvent 11 , in order to charge the solvent droplets. The charged solvent may thus be pneumatically driven by the gas flow 12 from the spray capillary onto the sample surface 20. The DESI sprayer 10 thus directs a spray of charged solvent droplets 13 onto the sample surface 20. Although an electrospray-type sprayer 10 has been described, it will be appreciated that various suitable devices that are capable of generating a stream of solvent droplets carried by a jet of nebulizing gas may be used to form the spray of (charged) solvent droplets 13. For instance, although Fig. 1 illustrates a DESI-MS interface, various similar solvent-driven ionisation interfaces have been developed and are known that operate according to similar physical principles to DESI and to which the techniques of the present invention may also be extended. For instance, by way of one example, Desorption ElectroFlow focussing ionisation ("DEFFI") sources may also suitably by used to generate the analyte ions.

Particularly, it is also contemplated that the solvent may not be charged in the sprayer device, as described above, but rather that the droplets of solvent may subsequently be activated or charged after their deposition onto the sample. For example, a voltage may be applied to the tissue section substrate to provide the charges.

In any case, the solvent droplets, whether charged or not, impact on and interest with the surface of the sample 20 in order to generate analyte ions. There are understood to be two main kinds of ionisation mechanism for DESI analyses, which may depend e.g. on the nature of the sample and the operating conditions of the DESI sprayer 10.

The first main ionisation mechanism is via a desorption process wherein the solvent droplets 13 hit the surface of the sample 20 and then spread out over a larger diameter and act to dissolve the analyte material with the dissolved analyte material then being released from the surface generating analyte ions as the solvent is evaporated. For example, the droplets may form a thin film of solvent on the surface of the sample 20 that desorbs the analyte molecules, and the desorbed analyte may then be released as secondary droplets by vaporisation or due to the impact of further solvent droplets on the sample. This may result in similar spectra to conventional electrospray ionisation ("ESI") techniques wherein primarily multiply charged ions are observed. It is believed that this mechanism leads to more multiply charged ions because multiple charges in the solvent droplets may easily be transferred to the desorbed analyte molecules. This mechanism may also be referred to as the "droplet pick-up" ionisation mechanism. This ionisation mechanism may be particularly suited for the ionisation and analysis of larger molecules such as peptides and proteins.

The second main ionisation mechanism is via direct charge transfer, either between a solvent ion and an analyte molecule on the surface of the sample 20; or between gas phase ions and analyte molecules on the surface or in the gas phase. This mechanism may be similar to what is observed in easy ambient sonic spray ionisation ("EASI") techniques, and typically generates only singly charged ions. This mechanism is normally observed for relatively smaller or lower molecular weight species compared to the desorption mechanism described above.

It will be understood that these techniques, including DESI, are generally "ambient" ionisation techniques. That is, the sample may be maintained and analysed under ambient or atmospheric conditions. Ambient ionisation ion sources such as DESI sources may further be characterised by their ability to generate analyte ions from a native or unmodified sample. For example, this is in contrast to other types of ionisation ion sources such as Matrix Assisted Laser Desorption Ionisation ("MALDI") ion sources that require a matrix or reagent to be added to prepare the sample prior to ionisation. It will be apparent that the requirement to add a matrix or a reagent to a sample impairs the ability to provide a rapid simple analysis of target material. Ambient ionisation techniques such as DESI are therefore particularly advantageous since firstly they do not require the addition of a matrix or a reagent and since secondly they enable a rapid simple analysis of target material to be performed. Ambient ionisation techniques such as DESI do not generally require any prior sample preparation or offline sample pre-treatment or separation. As a result, the various ambient ionisation techniques enable tissue samples to be analysed without necessitating the time and expense of adding a matrix or reagent to the tissue sample or other target material.

In other words, ambient ionisation techniques such as DESI may allow for a substantially direct analysis of the sample, i.e. without requiring any specific offline sample preparation or separation steps to be performed prior to the analysis. It will be appreciated that in the context of ambient ionisation the meaning of "direct" analysis is a well understood term of the art referring to in situ analyses performed directly from the surface of a sample. Direct analyses may thus avoid the need for any time-consuming sample separation or off line preparation steps e.g. using a matrix. Particularly, ambient ionisation techniques such as DESI may allow for samples to be directly analysed essentially in their native form. Naturally, this does not preclude any other steps that do not significantly alter the sample such as steps of washing the sample or mounting the sample. Furthermore, it is also contemplated that the sample may be treated with an enzyme such as protease in order to instigate digestion of the tissue, as explained further below, with the digested tissue then being analysed directly.

The embodiments described herein generally relate to methods of imaging a sample using mass spectrometry. In mass spectrometry imaging, the spatial distribution of the composition of sample may be visualised by analysing ions produced from multiple spatially separated regions of the sample. Thus, a method according to the techniques described herein may comprise surveying the surface of the sample by analysing multiple different regions of the sample in order to build up an image of the composition of the sample as a whole (i.e. including the multiple different regions). The sample 20 may thus be mounted on a moveable platform, and/or the DESI sprayer 10 may be moveable over the surface of the sample 20 so that the DESI sprayer 10 may probe or survey across the entire surface of the sample 20. In this way the DESI sprayer 10 is able to analyse ions that are localised at different regions of the sample in order to create a map or image of the distribution of constituents of the sample.

According to various examples, the sample 20 being analysed may comprise a tissue section such as a section of human or animal tissue. The sample 20 may thus be mounted on a slide 21 such as a glass, PTFE, or PTFE-coated glass slide, or some other suitably flat substrate. Human or animal tissue is generally composed inter alia of various peptides and proteins such as haemoglobins. It may be desirable, for various reasons, to be able to visualise the spatial distribution of these peptides and proteins within the tissue or tissue section. For example, mapping these compounds may help to elucidate the tissue type and/or disease state of the tissue being analysed. Thus, the DESI-MS (or other suitable) interface, may be used to visualise proteins and/or peptides directly from a tissue sample.

According to some examples, it is contemplated that the tissue sample (i.e. or tissue section) may be treated with an enzyme such as protease to digest the proteins into peptides. Particularly, the enzyme (protease) may be applied in situ onto the tissue sample so that the resulting peptides may be directly analysed from the tissue sample. It has been found that this may be done without delocalising the protein/peptide molecules, thus still providing meaningful results for mass spectrometry imaging. However, in other examples, intact proteins and/or native peptides within the tissue sample may be analysed directly without any prior enzymatic digestion.

It will be appreciated that the visualisation of complex proteins and peptides directly from a tissue sample is technically very challenging especially due to the large size and typical insolubility of these compounds combined with the potentially low abundance of these compounds (e.g. compared to background chemical noise and other constituents of the tissue). In order to achieve meaningful mass spectrometry imaging results for these compounds using a DESI-MS, or other solvent-driven ambient ionisation, technique, it is necessary to overcome these fundamental sensitivity issues. Accordingly, it has not previously been possible or practical to directly visualise proteins and peptides from tissue sections under ambient conditions, especially without any prior sample preparation such as a matrix. However, through extensive internal development, the Applicants have now developed techniques that are capable of visualising peptides and proteins directly from a tissue section using DESI-MS.

Although these techniques have been developed and substantially optimised for analyses of peptides and proteins, which suffer particularly from the problems mentioned above, it will be appreciated that the techniques may also generally be extended and applied to other molecules or biomolecules such as glycans.

According to the techniques described herein, various parameters or operating conditions of the DESI-MS process may be selected or controlled in order to enhance or substantially optimise the ionisation and analysis of the protein and peptide species of interest so as to allow a direct visualisation of these compounds from a tissue section. For instance, it has been found that by controlling one or more of the nebulizing gas pressure, the solvent flow rate, the voltage applied to the spray capillary, and the composition of the solvent, various improvements can be achieved in this respect allowing better visualisation of these compounds. Additional improvements may be achieved through a heating of the sampling capillary. It has also been found that washing the tissue section may provide further improvements without degrading or delocalising compounds from the surface of the sample. Particularly, the Applicants have developed techniques that promote the generation of multiply charged peptide and protein ions thus allowing a better visualisation of these compounds. It will be appreciated that each of the operating conditions of the DESI-MS analysis, as explained below, may be essentially independently controlled and optimisation of any of these conditions may by itself provide some improvement. However, it is believed that by controlling a plurality of these conditions, or controlling all of these conditions, in

combination, further improvements may be realised. For instance, it has been found that the combination of selecting an appropriate nebulizing gas pressure and solvent flow rate may help to promote larger droplets forming on the surface of the sample, thus promoting more efficient generation of multiply charged protein and peptide ions. Particularly, the formation of larger droplets may help to promote desorption-type ionisation processes (i.e. rather than a charge transfer-type ionisation process). Furthermore, the heating of the sampling capillary has been found to particularly help in enhancing the desolvation of such larger droplets. Thus, it will be appreciated that controlling various combinations of operating conditions may synergistically help to further improve the visualisation of peptides and proteins directly from a tissue section. It will also be appreciated that where one operating conditions has been substantially optimised, it may be possible to sacrifice another operating condition whilst still achieving a usable sensitivity for visualisation of peptides and proteins directly from a tissue section.

As mentioned above, whilst not essential, the ions may be separated using an ion mobility separator prior to their mass analysis. An ion mobility separator may be provided in order to better separate different classes of molecular ions. This may help to extract

(and hence be able to visualise) typically low abundance molecular species of interest from the high level of chemical noise inherent to DESI-MS tissue analysis. Furthermore, it has been recognised that DESI-MS tissue analysis may generate certain characteristic species that are not observed for other techniques such as MALDI. Particularly, it has been found that DESI-MS spectra include various peaks associated with compounds in the tissue that may be characterised as having relatively high mobility and charge states. Ion mobility separation may thus help to separate these characteristic species from the background noise. Fig. 2B shows a typical plot of mass to charge against drift time for a DESI-MS analysis of a rat liver tissue section. In order extract the multiply charged molecular species of interest from the high level of background noise due to singly charged clusters or solvent molecules, the visualisation may be restricted to a selected region, as shown in Fig. 2B, corresponding to ions having relatively high mobilities compared to other species of the same mass to charge ratio. Fig. 2A illustrates an improvement that may be achieved using ion mobility separation. The top panel shows a mass spectrum without any ion mobility separation where it can be seen that the peaks are generally swamped by background noise. The lower panel shows the spectra for the selected region shown in Fig. 2B, wherein the molecular peaks of interest are more apparent.

To generate the desired multiply charged proteins and peptides it has been found beneficial for the analyte species to be desorbed into relatively large solvent droplets.

However, this may in turn increase the amount of solvent that is passed to the ion analyser, increasing the background noise. Thus, it has been found that another improvement may be achieved by heating the sampling capillary. Whilst this is not essential, and at least some multiply charged ions may be detected without any such heating, these would typically comprise only relatively abundant proteins and peptides, and it has been found that heating the sampling capillary may improve the sensitivity with which these

compounds can be detected and/or may allow additional compounds to be visualised. The effect of heating the sampling capillary is illustrated in Fig. 3 which shows the mass spectral peaks for the rat liver tissue section described above within the selected mass-drift time region "A" shown in the lower panels (corresponding to the selected region of Fig. 2B). The illustrated peaks correspond to various haemoglobin ions of interest. As shown, the use of a heated capillary at a temperature of 430 ⁰ C, has been found to improve the overall sensitivity for detecting these compounds by a factor of approximately 3-5x.

Perhaps more significantly, it can be seen that the use of a heated sampling capillary also allows the detection of lower charge state (i.e. higher mass to charge ratio) proteins and peptides that are barely observable (if at all) without heating. It is believed that this is a result of a more efficient desolvation of the desorbed droplets from the tissue surface which may be particularly important for the analysis of proteins and peptides where the size of the desorbed droplets required to provide the desired multiply charged ions may be relatively large such that extensive desolvation may be required to allow the ions of interest to be reliably detected. Thus, with reference to Fig. 3, the spectrum obtained using a capillary heated at 430 ⁰ C shows a dramatically increased signal at higher mass to charge values compared to the spectra obtained with no sampling capillary heating (i.e. 25 ⁰ C), or moderate heating of the sampling capillary (of 215 ⁰ C). The central panel of Fig. 3 shows in finer detail the spectra for higher mass to charge values (within the mass to charge range from about 1400 to about 2000). These spectra were obtained for ions within the selected mass to charge-drift time region "B") shown in the lower panels.

Thus, in embodiments, the sampling capillary may be heated at or to a temperature above about 200 ⁰ C. Suitably, the sampling capillary may be heated at or to temperatures above about 250 ⁰ C, and more suitably above about 300 ⁰ C, or above about 400 ⁰ C. For example, the sampling capillary may suitably be heated at or to a temperature within the range of between about 300 and 1000 ⁰ C, such as between about 300 and 750 ⁰ C or between about 300 and 500 ⁰ C. More suitably, the sampling capillary may be heated at or to a temperature between about 400 and 1000 ⁰ C, such as between about 400 and 750 ⁰ C, between about 400 and 500 ⁰ C or between about 400 and 450 ⁰ C. For instance, the sampling capillary may be heated at or to a temperature of about 430 ⁰ C. The sampling capillary may be heated at or to temperatures greater than 500 ⁰ C. However, it will be appreciated that heating the sampling capillary at temperatures significantly higher than these ranges may not be practical. The optimal temperature within these ranges may depend on the sample and particularly on the ions of interest. For instance, the optimal sampling capillary temperature may vary within these broader ranges e.g. depending on whether the species of interest comprise large intact proteins or digested peptides.

One or more heaters or heating elements may be provided for heating the sampling orifice. For example, a heating jacket may be provided around an inlet or capillary leading into the sampling orifice. The heaters or heating elements may thus be arranged to heat the sampling orifice at (or to) the desired temperatures.

In embodiments, the sprayer device may also be heated.

The Applicants have further recognised that the nebulizer gas pressure (i.e. or gas velocity) may be controlled in order to promote the formation of relatively large solvent droplets on the sample surface which have been found to be advantageous in generating the desired multiply charged proteins and peptides of interest. The effects of changing the nebulizer gas pressure are illustrated in Figs. 4A and 4B. In both cases the same rat liver tissue section described above was analysed using a DESI-MS interface of the type shown in Fig. 1. The nebulizer gas was nitrogen. Fig. 4A illustrates the effect of the nebulizing gas pressure on the intensity of certain multiply charged haemoglobin peaks from the rat liver tissue section. Fig. 4B illustrates the effect of the nebulizing gas pressure on the intensity of the characteristic multiply charged high velocity species from the rat liver that are located within the selected region as shown (which essentially corresponds to the selected region shown in Fig. 2B). As shown, the nebulizing gas pressure may suitably be within the range from about 3 to 7 bar, such as from about 4 to 7 bar, or between about 5 to 7 bar, or most suitably between about 5 to 6 bar, where there is a slight optima for the peaks of interest. It is believed that the slight drop around 4 bar is an artefact of the specific sample being analysed.

It will be appreciated that these nebulizer gas pressures may be slightly lower than those that would typically be used for DESI-MS experiments. It is believed that the use of slightly lower pressures may help promote a desorption-type ionisation process (rather than a sonic spray-type ionisation process) wherein the solvent droplets remain in contact with the surface of the sample 20 for longer, allowing relatively larger droplets to form and promoting the generation of the multiply charged proteins and peptides of interest.

Similarly, the Applicants have recognised that the flow rate at which the solvent is introduced to the DESI sprayer 10 may be controlled so as to improve or optimise the generation of peptide and/or protein peaks of interest. The effect of the solvent flow rate is illustrated in Figs. 5A and 5B. Fig. 5A illustrates the effect of the solvent flow rate on the intensity of certain multiply charged haemoglobin peaks from the tissue section of rat liver. Fig. 5B illustrates the effect of the solvent flow rate on the intensity of the multiply charged high velocity species from the rat liver tissue section within the selected region as shown (which essentially corresponds to the selected region shown in Fig. 2B). As shown, solvent flow rates of about 1 μΙ_/ηιίη or greater, such as within the range of between about 1 and 4 μΙ_Ληίη, have been found to be particularly suitable, with solvent flow rates ranging between about 2 and 3 μΙ_Ληίη being most suitable. Depending on the sprayer design, these flow rates may be slightly higher than those that may typically be used in typical DESI-MS imaging experiments. Again, it is believed that these solvent flow rates may help promote the desorption-type ionisation process allowing relatively larger droplets to form and promoting the generation of multiply charged peptide and protein ions.

Thus, in embodiments, the nebulizer gas pressure and the solvent flow rate may each be controlled in order to promote ESI-type desorption of analyte from the sample surface promoting the formation of the desired multiply charged protein and/or peptide ions. That is, the operating conditions of the DESI sprayer 10 may be chosen so as to promote the formation of protein and/or peptide ions in order to increase the abundancy of these species so as to help overcome the inherent sensitivity issues associated with DESI- MS analysis of these species.

The composition of the solvent may also be selected so as to promote the generation of desired multiply charged peptide and protein ions.

Figs. 6A and 6B illustrate the effect of the solvent to water ratio wherein the solvent comprises aqueous acetonitrile with 0.2% by volume formic acid. Fig. 6A shows the effect on the intensities haemoglobin peaks whereas Fig. 6B shows the effect on the intensity of the multiply charged high velocity species from the rat liver i.e. those within the selected region shown in Fig. 2A. The nebulizing pressure used in each case was 5 bar. Generally, it has been found for acetonitrile/0.2% formic acid that a solvent to water ratio (by volume) within the range from about 50:50 to about 90: 10 provides suitable results, with the best results found with solvent to water ratios from about 50:50 to about 80:20, and more suitably between about 70:30 to about 80:20.

Figs. 7A and 7B illustrate the effect of the solvent to water ratio wherein the solvent comprises methanol with 0.2% formic acid. Fig. 7A shows the effect on the intensities haemoglobin peaks whereas Fig. 7B shows the effect on the intensity of the multiply charged high velocity species from the rat liver i.e. those within the selected region as shown (which essentially corresponds to the selected region shown in Fig. 2B). The nebulizing pressure used was 5 bar. Again, suitable results were achieved using solvent to water ratios (by volume) between about 50:50 to about 90: 10.

Figs. 8A and 8B compare the effects of acetonitrile and methanol as solvents. In all cases a fixed solvent to water ratio (by volume) of 80:20 was used. Fig. 8A shows the effect of the choice of solvent on the intensity of the multiply charged haemoglobin peaks from the rat liver sample. Fig. 8B shows the effect of the solvent on the intensity of the multiply charged high velocity ion mobility species peaks from the rat liver from the selected region as shown. In general, it was found that both solvents are suitable.

However, it has been found that acetonitrile may provide slightly more intense protein and peptide peaks than methanol. Methanol is the solvent that is typically used (typically at a concentration of about 95-98% methanol) for conventional DESI-MS imaging experiments. It will be appreciated that various other electrospray compatible solvents, such as ethanol or isopropanol, may also suitably be used, at varying solvent to water ratios. For example, other suitable solvents may include dichloromethane (optionally mixed with methanol), dichloroethane, tetrahydrofuran, ethanol, propanol, methanol, nitromethane, toluene (optionally mixed with methanol or acetonitrile), and water.

It has been found that concentrations of between about 0.2 and 0.4% formic acid may be appropriately used for the analysis of proteins and/or peptides (in combination with either of acetonitrile or methanol). It will be appreciated that various other electrospray compatible acids, such as acetic acid, may also suitably be used. It is also contemplated that various additives may be used within the solvent in order to enhance the generation of multiply charged species and/or to change the charge state composition. For example, super charging agents such as dimethyl sulphur oxide (DMSO), 3-nitro benzyl alcohol (3-NBA) or sulfolane may be introduced into the solvent. The effect of 3-NBA concentration is shown in Figs. 9A and 9B. For 3-NBA, it has been found that best results can be achieved using concentrations between about 0.01 and 0.5%, and more particularly between about 0.25 and 0.5%. Above this, it was found that there was some suppression of the peaks of interest. The effects of DMSO are illustrated in Figs. 10A and 10B. Concentrations of DMSO between about 0.01 and 0.5% were found to be suitable.

Other suitable additives may include volatile salts or buffers such as ammonium acetate or ammonium bicarbonate. Various other additives including dimethylformamide (DMF), trifluoroacetic acid, heptafluorobutyric acid, sodium dodecyl sulphate,

ethylenediaminetetraacetic acid, and involatile salts or buffers such as sodium chloride and sodium phosphates may also be tolerable in small amounts.

Another parameter that may be controlled is the voltage applied to the DESI sprayer to charge the solvent droplets. Figs. 1 1 A and 11 B illustrate the effect of changing the voltage applied to the spray capillary of the DESI sprayer device. As shown, capillary voltages above about 4 kV may be particularly suitable, such as capillary voltages between about 4 and 5 kV, such as voltages of about 4.5 kV.

It has also been discovered that the washing of the tissue sample may help to promote the peaks of interest without altering the composition of the tissue sample (i.e. without degrading or denaturing the proteins within the tissue). The effect of the washing is illustrated in Figs. 12A and 12B. Fig. 12A shows the effects of the washing on the detection of the haemoglobin peaks within the rat liver tissue sample whereas Fig. 12B shows the effects on the detection of the characteristic high mobility multiply charged molecular species from the rat liver tissue sample within the selected region shown in Fig. 2B. Washing the tissue section with ethanol may provide some improvements. Further improvements may be achieved by washing the tissue section with ethanol and chloroform. Particularly, the washing procedure may comprise a series of ethanol/water washes followed by a final chloroform washing step. For example, the tissue section may consist of the series of ethanol/water washes comprising (by volume): 70/30% for 1 minute;

90/10% for 1 minute and 100/0% for 1 minute followed by a final washing step in 100% chloroform for 25 seconds. Figs. 12A and 12B show that the combination of ethanol and chloroform washes may provide more intense protein and peptide peaks than the use of ethanol alone, or for no washing at all (as would typically be the case for DESI-MS analyses). The spectra shown in Figs. 12A and 12B also confirm that the washing process does not significantly alter the composition of the tissue sample. Thus, the tissue sample is not digested or denatured by the washing procedures and the tissue molecules of interest are not delocalised on the surface. Thus, the washes may remove lipids or impurities from the surface, but do not disrupt the proteins and peptides of interest. Each of the experiments described above were carried out on a Waters SYNAPT HDMS G2-Si Q-TOF instrument equipped with a 2D DESI stage available from Prosolia, Inc. of Indianapolis. When the DESI stage was mounted, the electrospray inlet block was installed along with a sampling capillary that was adapted with a heating jacket for heating the sampling capillary. For the experiments described above, where one parameter is being investigated, the other parameters were naturally held constant. The experiments were all carried out directly from a section of fresh frozen rat liver.

Except where that parameter was being investigated, the respective values used for each of the experiments were as follows: Capillary voltage = 5 kV; Nebulizing gas

(nitrogen) pressure = 4 bar; Solvent composition (ACN:H ₂ 0) = 80:20 with 0.2% formic acid; Solvent flow rate = 4 μΙ_/ηιίη; Sampling Capillary temperature = 430 ⁰ C.

Similar results were obtained from analysing a full tissue section of mouse brain. For instance, through DESI-ion mobility-MS imaging of a mouse brain it was possible to observe several trend lines in the mass to charge ratio to drift time plots corresponding to various native intact peptides/proteins of interest. The results of this analysis are shown in Figs. 13A and 13B which show the mass spectral peaks obtained within the indicated selected regions of mass to charge-drift time. The steepest gradient was found to correspond to multiply charged (of up to +8) endogenous peptides. Another trend line was observed with a lower gradient corresponding to highly multiply charged proteins (of up to +24, Fig. 13A), with the most abundant species identified as being an isoform of the myelin basic protein. The lowest gradient trend line corresponded to small number of remaining singly charged lipids that separated from the background of cluster or solvent ions (Fig. 13B).

Figs. 14A and 14B show the results of further experiments, using the same experimental conditions mentioned above, on fresh frozen mouse liver tissue section. In this case, again, several distinct trend lines were observed in the mass to charge to drift time plots corresponding to various native intact peptides/proteins of interest. As shown in Fig. 14A, it was found that the protein signals were more intense and these were identified to be related to four haemoglobin chains (alpha and beta). Fig. 14B shows the DESI imaging results for a different selected mass to charge to drift time region.

Fig. 15 shows further experimental data obtained from a fresh frozen mouse brain tissue section using DESI-MS wherein a solution of trypsin was applied on site to the tissue section using a SunCollect automated spraying device (available from SunChrom GmbH) and the tissue was then incubated for four hours at 37 ⁰ C prior to the DESI-MS analysis. The tryptically digested peptides were then directly analysed as described above. The ions associated with the trendline with the highest ion mobility values correspond to doubly and triply charged peptides. The second trendline was found to correspond to highly multiply charged ions of an isoform of the myelin basic protein, the third trendline corresponds to singly charged peptides, and the slowest trendline corresponds to remaining singly charged lipids. The doubly charged tryptic peptides were distributed to specific parts of the brain tissue, demonstrating that the different steps of the sample preparation process did not alter the location of proteins across the tissue section. It will be appreciated that the data presented above was obtained in a positive mode. However, similar results have also been obtained in the negative mode. In the negative mode it was generally found to be better to not add any acid to the solvent.

As mentioned above, various ion analysers may suitably be used to analyse the ions generated according to the techniques described herein. In embodiments, the ion analyser may comprise a hybrid ion mobility-mass spectrometers which has been found to be particularly suitable for analysing the data obtained from a tissue sample using the DESI (or similar) source. Furthermore, various known techniques for manipulating the ions and/or processing the resulting data may advantageously be used to further improve the analysis.

For example, the peptide and protein ions generated according to the techniques described herein are typically multiply charged. In fact, often, when attempting to visualise proteins and peptides from a tissue the only ions of interest are multiply charged. Thus, in embodiments, a quadrupole mass filter may be provided and arranged in synchronisation with an ion mobility separator device to reject the singly charged ions resulting in a decrease in the processing requirements. Furthermore, in some embodiments, where an ion mobility separator device is provided, knowledge of the ion mobility (i.e. or drift times) of the ions may be used to determine or estimate the charge state of the candidate ion and optionally include or reject the ion from the MS/MS experiments. This information may e.g. then be used to set an appropriate collision energy (or fragmentation conditions) based upon this estimated charge state and its mass to charge ratio value thus removing the need to de-isotope the survey spectra thereby reducing the processing required. The ion mobilities (i.e. or drift times) and mass to charge ratios of the candidate ions may also be used to determine the optimal fragmentation methodology employed i.e. whether the candidate ion is fragmented using ETD or CID. To further reduce processing requirements data may be discarded from areas that contain charge states or information that is not of particular interest. For example, the drift time may be used to identify and select only a certain class of compounds such as lipids or glycans, polymers such as PEG or PPG, or pharmaceuticals containing active ingredients that are reacted with polymers such as PEG to increase their "lifetime" in the body. Compounds may also be selected if they fall upon a given or known drift time - mass to charge ratio trend line and select the appropriate collision energy for the mass to charge ratio within that class. Such techniques may be implemented as described in WO 2014/0140542 (MICRO MASS).

Furthermore, in embodiments the ion analyser may comprise a mass spectrometer including a fragmentation or reaction device. The ion analysis may thus include steps of fragmenting or reacting at least some of the ions generated by the DESI source to form product or daughter ions, wherein the product or daughter ions are then generated. The product or daughter ions so generated may be highly characteristic and may help to facilitate identification and/or confirmation of the presence of proteins and/or peptides within the tissue sample. For instance, the mass spectrometer may be operated in a first mode wherein the protein and/or peptide parent ions of interest are analysed directly and a second mode wherein the protein and/or peptide ions generated from the tissue sample are caused to react or fragment and wherein the resulting product or fragment ions are analysed. In embodiments, the mass spectrometer may be arranged to obtain one or more parent ion mass spectrum and one or more product or fragment ion mass spectrum for each location on the tissue sample that is being visualised. In embodiments the mass spectrometer may be arranged to repeatedly and alternatively obtain such spectra in order to generate a series of parent ion and product ion mass spectra. These techniques may facilitate processing of the complex data obtained from the DESI-MS analysis of the tissue sample. In embodiments a series of product or fragment ion mass spectra may be obtained at a plurality of different collision or reaction energies.

Although the present invention has been described with reference to various embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made without departing from the scope of the invention as set forth in the accompanying claims.