WO2011073740A1

WO2011073740A1 - Matrices for mass spectrometry imaging

Info

Publication number: WO2011073740A1
Application number: PCT/IB2009/056037
Authority: WO
Inventors: Isabelle Fournier; Michel Salzet; Céline MERIAUX; Julien Franck
Original assignee: Centre National de la Recherche Scientifique CNRS; Universite Lille 1 Sciences et Technologies
Current assignee: Centre National de la Recherche Scientifique CNRS; Universite Lille 1 Sciences et Technologies
Priority date: 2009-12-15
Filing date: 2009-12-15
Publication date: 2011-06-23
Anticipated expiration: 2012-06-15

Abstract

Liquid ionic matrices for MALDI Mass Spectrometry Imaging (MALDI-MSI) of tissue sections and their use for the detection and localization of analytes such as lipids. The matrices comprise 2,5- dihydroxy benzoic acid and an organic base so as to reduce delocalization of analytes.

Description

MATRICES FOR MASS SPECTROMETRY IMAGING

The present invention concerns new ionic liquid matrices and their use in mass spectrometry imaging.

MALDI Mass Spectrometry Imaging (MALDI-MSI) uses the detection capability of mass spectrometry with the positional information of molecular histology, generating mass spectra correlated to known locations within a tissue. MALDI mass spectrometry imaging is able to reveal the distribution of a large range of analytes. This information can be used to determine the distribution of an analyte throughout a tissue or organism. Since its introduction by the team of Caprioli Mass Spectrometry Imaging (MSI) has become a powerful and versatile tool for analyzing different classes of endogenous and exogenous molecules including drugs imaging^2"4, peptides and polypeptides^5"9. Lipids were widely studied by MSI^10"14 due to the fact that this class of biomolecules is extremely important and is involved in different pathology including Alzheimer disease¹⁵ and down syndrome¹⁶.

However, one of the major problems with MALDI-MSI is the sample preparation and the matrix. Some analytes, such as lipids and drugs, are highly mobile during tissue preparation and are soluble in the solvent that is used for matrix preparation, resulting in substantial derealization of the analyte.

Lipid analysis by MALDI-MS is generally performed using 2,5-Dihydroxybenzoic

Acid (2,5-DHB) as matrix due to the fact that this matrix presents few peaks in the lower m/z, a very good stability under vacuum and analysis in both positive and negative mode are possible^17"19. However, 2,5-DHB characteristically crystallises in the shape of needles that grow from the outer rim of the deposited solution. In addition, a fine poly crystalline film forms in the inner part with salts species accumulating in this region despite reduced crystal size and reduced heterogeneity compared to classical MALDI sample preparation. 2,5-DHB is also known for presenting hot spots. This phenomenon can be overcome in classical MALDI by carefully and manually moving the sample under laser irradiation towards the needle part of the crystals. However, due to the spatial resolution required in MALDI MSI this is to a large extend not possible. To avoid this problem, Murphy and colleagues ¹⁴ have proposed sublimation of 2,5-dihydroxybenzoic acid without the use of a solvent that dissolves the lipid in the tissue.

2,6-dihydroxyacetophenone (2,6-DHAP) as matrix was employed for analysis of lipids in both positive and negative mode^19"21. However, 2,6-DHAP was shown to be unstable under vacuum leading to the sublimation of the matrix 30 or 45 min after introduction of the sample into the mass spectrometer ²². Others substances were tested as matrices for lipid analysis including p-nitroaniline (PNA). PNA seemed to be a good alternative for lipid detection but instability under vacuum was also observed for this matrix ²³. 6-aza-2-thiothymine (ATT) was proposed as a good stable matrix for lipid analysis with interesting properties for phospholipid detection ²⁴. Recently, 2- Mercaptobenzothiazole (MBT)²⁵ was shown to be very efficient for lipid detection after spray deposition providing very homogenous crystallization, a high stability under vacuum and allowing the detection of a wide variety of lipids.

For some classes of lipids, the incorporation into matrix crystals is not necessary for their detection in gas phase. Different procedures to apply matrix on tissue including solvent-free matrix coating ²⁶ for phospholipid detection and matrix sublimation²⁷ were developed to enhance crystal formation on the tissue avoiding delocalisation of analytes.

Another alternative, is the use of Ionic matrices (IM) as we have already described for peptides²⁸. IMs were presented as a good alternative to conventional matrices for MSI for their very good stability under vacuum and the better sensitivity often observed ²⁹. Recently, the use of Liquid Ionic Matrix (ILM) for detection of gangliosides form mouse brain was described ³⁰. ILM was sprayed over the tissue preventing the analysis of gangliosides in reflector mode due to the fact that generally spray deposition involves the deposition of very small droplets of matrix solution leading to low extraction efficiency.

A good compromise between extraction of analytes from the tissue and derealization may be achieved by using microspotting of matrices. Due to the amount of solution deposited on the surface of the tissue, good extraction efficiency is achieved leading to the delocalization of analytes only on the surface of the spots.

Sample preparation is a very important parameter in Mass Spectrometry Imaging experiments. The choice of the matrix and the procedure is critical to obtain high quality spectra and molecular images.

For the detection of lipids and of other analytes in MSI, the conventional 2,5-DHB matrix is commonly used but heterogeneous crystallization is caused by the microspotting of the matrix. It was shown that surface properties of tissues could be modified by various treatments and thus crystallization of 2,5-DHB could be greatly improved. However, lipids and other analytes are known to be very soluble in a wide variety of solvents including alcohol, acetone, chloroform or xylem. It is thus very difficult to submit tissue sections to specific washing/treatment steps without losing some molecules of interest.

To overcome this drawback 2,6-DHAP as matrix was tested. The crystals obtained after microspotting were more homogeneous than those observed with 2,5-DHB microspotting even without performing any chemical treatments. However, this matrix is known to be unstable under high vacuum and the results presented herein show similar observations after less than lh. Sublimation of 2,6-DHAP occurred in the MALDI source and therefore 2,6-DHAP is not compatible with MSI. In fact, following the size of the tissue section and the resolution, the time required for analysis could be reaching more than 10 hours depending on the instrument used.

The present application describes new liquid ionic matrices (IML) and their use for MALDI mass spectrometry imaging. These matrices are obtained by adding a base to 2,5- DHB. These matrices enable a very stable deposition of the matrix leading to the formation of very homogeneous spots of matrix. Moreover, the matrices of the present invention are very stable under vacuum and the spectra obtained were similar to those obtained using conventional 2,5-DHB or 2,6-DHAP as matrix in terms of the species which are detected. Further, the matrices of the present invention are efficient in both positive and negative mode. Another advantage is that a low amount of matrix is required to obtain spectra with intense peaks. The matrices of the present invention are therefore very suitable for the detection of various analytes including lipids in MSI experiments after microspotting. 2,5 DHB /3AP is a matrix of choice for the detection of lipids for MSI experiments after microspotting.

The liquid ionic matrices of the present invention are especially useful for the quantification of small molecules and lipids in tissue sections. Upon microspotting of the matrix onto the tissue section, most of the analyte is locally extracted from the tissue section and accumulates in the microspot of matrix where it the analyte can be quantified efficiently by MALDI-MSI. SUMMARY OF THE INVENTION

The present invention relates to methods for detection and/or quantification of an analyte in a biological tissue section comprising the following steps: a) Depositing a liquid ionic matrix onto the biological tissue section wherein the liquid ionic matrix comprises 2,5-DHB and an organic base, b) Analysing the biological tissue by Matrix Assisted Laser Desorption Mass spectrometry Imaging.

In preferred embodiments, the liquid ionic matrix is selected from, 2,5-DHB/ ANI,

2,5-DHB/3AP, 2,5-DHB/Pyr, 2,5-DHB/DANI and/or 2,5-DHB/DEANI.

Preferably, the liquid ionic matrix is deposited onto the biological tissue section by micro-spotting.

In preferred embodiments, at least one micro-spot of between 5 and 15 nL of ionic liquid matrix is deposited onto the biological tissue section.

Preferably, a defined pattern of micro-spots of ionic liquid matrix is deposited onto the biological tissue section.

Advantageously, the analyte is a lipid. Even more advantageously, the analyte is selected from phospoethanolamines (PE), phosphocho lines (PC), phosphoserines (PS), sphingomyelins, phospho inositols (PI), sulfatides (ST), hydroxylated sulfatides (ST-OH). Most preferred, the analyte is a small molecule having a size comprised between 23 and 2000 M/z.

In preferred embodiments, the biological tissue section is analysed by Matrix Assisted Laser Desorption Mass spectrometry Imaging in both positive and negative reflector mode.

Preferably, the analyte is both detected and quantified in the biological tissue section.

Another object of the present invention is a composition comprising between 5 and 30 mg/mL 2,5-DHB in a solvent and an organic base selected from 3AP, ANI, Pyr, DANI and/or DEANI.

In preferred embodiments, the ratio organic base/2,5-DHB is comprised between 0,5 and 2.

Preferably, the solvent comprises methanol, ethanol and TFA.

The present invention is also related to the use of such a composition for the detection and/or quantification of an analyte in a biological tissue section by Matrix Assisted Laser Desorption Mass spectrometry Imaging.

In preferred embodiments, the present invention is related to the use of a composition for detection and/or quantification of an analyte in a biological tissue section by Matrix Assisted Laser Desorption Mass spectrometry Imaging wherein the analyte is a lipid.

DETAILED DESCRIPTION OF THE INVENTION

Mass spectrometry is an analytical tool used for measuring the molecular mass of an analyte. Mass spectrometers can be divided into three fundamental parts, namely the ionization source, the analyzer and the detector.

One of the ionization methods used for the majority of biochemical analyses is MALDI (matrix assisted laser desorption ionization). MALDI is based on bombardment of sample molecules with a laser light to bring about sample ionization. A MALDI matrix is required to protect the sample and to facilitate ionization and desorption. The analyte or sample is embedded in this matrix.

Any analyser may be used in the methods of the present invention. Time-of- flight mass spectrometry is a common technique largely described in the litterature. TOF-MS is a method of mass spectrometry in which ions are accelerated by an electric field. The velocity of the ion depends on the mass-to-charge ratio. The time that it subsequently takes for the particle to reach a detector at a known distance is measured. This time will depend on the mass-to-charge ratio of the ion. From this time and the known experimental parameters one can find the mass-to-charge ratio of the ion.

In the methods of the present invention, the type of mass spectrometer advantageously used with MALDI is the TOF (time-of- flight) mass spectrometer. However, any suitable mass spectrometer may be used in the methods of the present invention.

The present invention relates to MALDI mass spectrometry imaging (MALDI-

MSI). MALDI-MSI links the universal detection capability of mass spectrometry with the positional information of molecular histology, generating mass spectra correlated to known locations within a biological tissue. In MALDI imaging, tissue sections are mounted onto a support and a suitable MALDI matrix is applied to the tissue. The support is inserted into a MALDI mass spectrometer which records the spatial distribution of different analytes. Suitable image processing software is used to import data from the mass spectrometer to allow visualization of analytes on the optical image of the tissue section.

A MALDI-MSI process comprises two steps: preparation of the sample including deposition of the matrix followed by ionization and desorption of the analyte by intense short pulses of laser light. The matrix provides for absorption of energy from the laser light to desorb the analyte and promotes ionization.

The term "matrix" refers to a material which generates matrix-embedded analyte molecules (i.e. proteins) that are successfully desorbed by laser irradiation and ionized from the solid phase. The matrix usually consists of molecules of low molecular weight to provide for vaporization but large enough not to evaporate during sample preparation or while standing in the spectrometer. Matrices also have a strong optical absorption in the UV range, so that they rapidly and efficiently absorb the laser irradiation. In classical MALDI, the matrix solution is mixed with the sample or applied onto the sample. The organic solvent allows hydrophobic molecules to dissolve into the solution, while the water allows for water-soluble (hydrophilic) molecules to do the same. Then, the solvents vaporize, leaving only the re-crystallized matrix, but now with analyte spread throughout the crystals. The matrix and the analyte are said to be co-crystallized in a MALDI spot.

A major problem in MALDI-MSI is the derealization of analytes, especially of lipids and small molecules such as drugs, upon application of the MALDI matrix onto the tissue section.

The present invention is related to new MALDI-MS matrices and to their use for the detection of analytes. These MALDI-MSI matrices provide for less derealization of analytes and crystallize homogeneously onto the tissue sample.

The present invention relates to methods for detection and/or quantification of an analyte in a biological tissue section comprising the following steps:

a) Depositing a liquid ionic matrix onto the biological tissue section wherein the liquid ionic matrix comprises 2,5-DHB and an organic base, b) Analysing the biological tissue by Matrix Assisted Laser Desorption Mass spectrometry Imaging.

The methods of the present invention are preferably in vitro methods.

These methods can be used to localize and identify analytes particularly lipids and small molecules throughout a tissue or organism under different experimental or therapeutic conditions.

The methods of the present invention provide for detection of an analyte in a biological tissue section. The term "detection" refers both to localization and identification of the analyte in the tissue section. Further, the methods of the present invention may provide for quantification of the analyte in the biological tissue section.

Any analyte and more particulary any biological analyte may be detected with the methods of the present invention. In preferred embodiments, the analyte is a lipid. Lipids are a broad group of naturally-occurring molecules which includes fats, waxes, sterols, monoglycerides, diglycerides, phospholipids, etc. Lipids may be broadly defined as hydrophobic or amphiphilic small molecules.

In preferred embodiments, the analyte is selected from phospoethanolamines (PE), phosphocho lines (PC), phosphoserines (PS), sphingomyelins, phosphoinositols (PI), sulfatides (ST), hydroxylated sulfatides (ST-OH).

In other embodiments, the analyte is a small molecule having a size comprised between 23 and 2000 M/z, preferably between 23 and 500, 1000 or 1500 M/z. M/z refers to mass-to-charge ratio obtained by dividing the mass of an ion by the unified atomic mass unit and by its charge number.

Such small molecules may be subject to derealization in biological tissue sections when a MALDI matrix is applied. Advantageously, the matrices of the present invention provide for detection of such molecules without derealization within the biological tissue section. Small molecules of interest may for example include various therapeutic molecules i.e. drugs.

The analyte may be detected, localized and/or identified in any biological tissue section of interest. The biological tissue section preferably has a thickness comprised between 5 and 20μιη. Any tissue or organism may be analyzed in the methods of the present invention. The tissue section is prepared according to known methods. Thin sections/slices are typically obtained from frozen tissues and applied onto solid supports. The tissue sections are then usually dried in a dessicator. The tissue sections immobilized onto a solid support may also be washed prior to further preparation of the sample for MALDI-MS. Solid supports or plates suitable for MALDI-MS are largely described in the literature. Any suitable support may be used in the methods of the present invention.

The liquid ionic matrix is deposited onto the biological tissue section using any suitable method.

The matrix may for example be dispensed by pipetting, by spraying or by printing small amounts of matrix by a chemical injet printer. Printers and spotters which deposit a small amount of MALDI matrix in a user-defined pattern directly onto the tissue by so called "microspotting" have been described. Deposition of the matrix in small amounts on a defined spot by micro-spotting is preferred in the methods of the present invention to further avoid any derealization of the analyte to be detected in the biological tissue section.

In preferred embodiments, at least one micro-spot of between 5, 10 and 15 nL of ionic liquid matrix is deposited onto the biological tissue section.

Thereafter, the Mass spectrometer records, detects and identifies each analyte in each microspot. Suitable software is used to allow visualization and comparison with the optical image of the biological tissue section.

The liquid ionic matrices of the present invention comprise an acid (2,5-DHB 2,5- Dihydroxybenzoic Acid) and an organic base. The organic base is preferably selected in the group consisting of 3-Acetylpyridine (3AP), Pyridine (Pyr), Aniline (ANI), N,N- dimethylaniline (DANI) and Ν,Ν-diethylaniline (DEANI).

In preferred embodiments, the liquid ionic matrix is selected from, 2,5-DHB/ ANI, 2,5-DHB/3AP, 2,5-DHB/Pyr, 2,5-DHB/DANI and/or 2,5-DHB/DEANI.

The biological tissue section may be analysed by Matrix Assisted Laser Desorption

Mass spectrometry Imaging in positive ion and/or negative ion reflector mode.

Preferably, the solvent comprises methanol, ethanol and TFA. The present invention is also related to the use of such a composition as matrix for the detection and/or quantification of an analyte in a biological tissue section by Matrix Assisted Laser Desorption Mass spectrometry Imaging.

In preferred embodiments, the present invention is related to the use of such a composition as matrix for detection and/or quantification of an analyte in a biological tissue section by Matrix Assisted Laser Desorption Mass spectrometry Imaging wherein the analyte is a lipid.

Another object of the present invention is a method for the in vitro clinical diagnosis on a biological tissue section using a matrix or the methods according to the present invention.

FIGURES

Figure 1: Optical images of spots saved after deposition of (a) 2,5-DHB, (b) DHAP and (c) 2,5-DHB/3AP on a rat brain tissue section.

Figure 2: MS spectra acquired in positive reflector mode after deposition of DHAP and saved after (a) introduction of the target in the mass spectrometer and (b) after lh in the vacuum. MS spectra acquired positive reflector mode after deposition of 2,5-DHB/3AP and saved after (c) introduction of the target in the mass spectrometer and (d) after 2 days in the vacuum.

Figure 3: Structure of ILM including (a) 2,5-DHB/ANI (Aniline), (b) 2,5-DHB/DANI (Ν,Ν-dimethylaniline), (c) 2,5-DHB/DEANI (Ν,Ν-diethylaniline), (d) 2,5-DHB/Pyr (Pyridine) and (e) 2,5-DHB/3AP (3-acetylpyridine).

Figure 4: MS spectra acquired in positive reflector mode after deposition of (a) 2,5- DHB/ANI, (b) 2,5-DHB/DANI, (c) 2,5-DHB/DEANI, (d) 2,5-DHB/Pyr and (e) 2,5- DHB/3AP.

Figure 5: MS spectra acquired in negative reflector mode after deposition of (a) 2,5- DHB/ANI, (b) 2,5-DHB/DANI, (c) 2,5-DHB/DEANI, (d) 2,5-DHB/Pyr and (e) 2,5- DHB/3AP.

Figure 6: MS spectra acquired in positive reflector mode after deposition of (a) 2,5-DHB and (b) 2,5-DHB/3AP. MS spectra acquired in negative reflector mode after deposition of (c) 2,5-DHB and (d) 2,5-DHB/3AP.

Figure 7: MALDI mass spectra from Olanzapine (lOmM) obtain in positive reflector mode with the ILM 2,5-DHB/ANI EXAMPLES

Materials

2,5-Dihydroxybenzoic Acid (2,5-DHB), 2,6-dihydroxyacetophenone (2,6-DHAP), 3- Acetylpyridine (3AP), Pyridine (Pyr), Aniline (ANI), Ν,Ν-dimethylaniline (DANI), N,N- diethylaniline (DEANI), Trifluoracetic acid (TFA), Ethanol (EtOH), Water Chromasolv plus for HPLC (H₂0) were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France). Samples

Rat brains.

Adult male Wistar rats weighing 250-35 Og (animal use accreditation by the French ministry of the agriculture N° 04860) maintained under standard care were used. Animals were sacrificed by decapitation and immediately dissected to remove the brain.

Rat brain cryosections

Frozen rat brains were cut into 10

tissue sections using a cryostat Leica CM1510S (Leica Microsystems, Nanterre, France). The sections were applied onto ITO- coated conductive glass slides (Bruker Daltonics, Bremen, Germany) and dried in a desiccator for 10 minutes.

Matrix preparation

Preparation of the 2,6-dihydroxyacetophenone (2,6-DHAP) matrix

20 nL of solution containing 20 mg/mL of 2,6-DHAP in EtOH/aqueous TFA 0.1% (7:3, v:v) were deposited following the same procedure than 2,5-DHB deposition.

Preparation of the 2,5-Dihydroxybenzoic Acid (2,5-DHB) matrix

20 nL of solution containing 20 mg/mL of 2,5-DHB in EtOH/aqueous TFA 0.1% (7:3, v:v) were deposited using a high accurate position Chemical Inkjet Printer CHIP- 1000 (Shimadzu, Kyoto, Japan).

Preparation of the Liquid ionic matrices

Liquid ionic matrices 2,5-DHB/ANI, 2,5-DHB/DANI, 2,5-DHB/DEANI, 2,5-

DHB/Pyr and 2,5-DHB/3AP were used as matrix and were prepared just prior to use by adding 1.2 equivalent of organic base to a solution containing 20 mg/mL of 2,5-DHB (1 equivalent) in EtOH/aqueous TFA 0.1%> (7:3, v:v). Solutions were then vortexed/agitated for several minutes before use. Matrices solutions must be used within one day of preparation. The total volume of ILM was set to 5 nL and was deposited directly on tissue using the CHIP- 1000.

Mass Spectrometry Imaging of lipids

Molecular images were acquired using an UltraFlex II MALDI-TOF/TOF instrument

(Bruker Daltonics, Bremen, Germany) equipped with a Smartbeam laser with a repetition rate up to 200 Hz that was controlled by FlexControl 3.0 (Build 158) software (Bruker Daltonics, Bremen, Germany). Images were obtained in both positive and negative reflector modes, and MALDI MS spectra were acquired in the 300-2000 mlz range. A total of 500 spectra were acquired at each spot at a laser frequency of 200 Hz. The images were saved and reconstructed using Flexlmaging 2.1 (Build 15) (Bruker Daltonics, Bremen, Germany).

2,5-DHB deposition.

Due to the heterogeneous crystals distribution, 2,5-DHB was deposited on a discrete location of a rat brain tissue section using a microspotter in order to improve the crystallisation. In fact, it was shown that the use of microspotter leads to the formation of very homogeneous spots of matrices as presented with 3-hydroxypicolinic acid (3HPA)³¹ known for the formation of heterogeneous crystals and "hot spots". For these reasons, the first tests to detect lipids directly from tissue section were realized after deposition of 2,5- DHB using the microspotter. During the deposition, stable printing was observed leading to the formation of heterogeneous spots as presented Figure la. Crystallization of 2,5- DHB with the shape of a circle was observed leading to a lack of information in the center of the spots. It could be a strong drawback by considering that position teaching for MALDI-MSI experiment after microspotting, is generally done using the center of spots. Laser will thus shot in the center where no crystals are observed and therefore no analytes will be detected. The formation of such crystals could be explained by physical properties of tissue section without any chemical treatments. In fact, matrix solutions for lipid analysis consist generally to the use of polar solvents such as alcohol including EtOH. By using these solvents, droplets of matrix solution spread on the surface of the tissue leading to the formation of 2,5-DHB crystals from the outer rim of the deposited solution. After chemical treatments using a chloroform bath, properties of tissue become more hydrophobic than no treated samples. The 2,5-DHB spots are therefore more homogenous (data not shown) in such case proving that crystals formation of 2,5-DHB after microspotting could be influenced by the properties of the tissue. However, chemical treatments are not recommended in the case of lipids analysis due to the fact that many lipids can be removed by chloroform or alcohol.

2,6-DHAP deposition

Another matrix was then tested in same conditions than 2,5-DHB in order to find a suitable matrix for MSI of lipids after microspotting. 2,6-DHAP was then deposited even if this matrix is known to be unstable under high vacuum. The printing was stable during the deposition leading to the formation of more homogenous spots of 2,6-DHAP than those observed with 2,5-DHB deposition (Figure lb). The first spectra acquired just after the introduction of the target in the MALDI-TOF, show very intense peaks from lipids in the 600 to 900 m/z mass range (Figure 2a). 2,6-DHAP seems to be very efficient to lipids detection but after lh in the source vacuum, no peaks from lipids were detected anymore (Figure 2b). These results are in good agreement than those previously presented ²² and confirmed that 2,6-DHAP in really unstable under high vacuum and this phenomenon is clearly increased after matrix deposition or if the amount of matrix deposited is very low. Although 2,6-DHAP is an effective matrix for direct lipids analysis, this matrix is not adapted for MSI of lipids because of the time needs to raster the whole tissue section with the laser which usually exceed lh ³².

ILM deposition

In order to overcome the drawback induced by the heterogeneous crystallization of 2,5- DHB or the instability of 2,6-DHAP under high vacuum, the use of IM was investigated. It was shown that the use of IM has greatly improved the detection of analytes for MSI experiments ²⁸' ²⁹. Concerning lipids analysis, the ILM based on the use of HCCA (Alpha- cyano-4-hydroxycinnamic acid) was presented as a good matrix to enhanced gangliosides directly from tissue section. 2,5-DHB is a strong matrix for lipids analysis and different teams have shown that ILM based on 2,5-DHB were effective for analysis of

33 35 36

oligosaccharides ^" and phospholipids . These matrices were presented as very homogeneous matrices due to the fact that no crystals formation was observed. This homogeneity allows quantification studies by MALDI-TOF ³⁷ made possible by the great analytes distribution over the matrix spots. ILM including 2,5-DHB/ANI, 2,5-DHB/DANI, 2,5-DHB/DEANI, 2,5-DHB/Pyr and 2,5-DHB/3AP as matrix (Figure 3) were then studied and were deposited on a discrete location of a rat brain tissue section using the microspotter. Very stable and reproducible printing was observed during the deposition for all ILM because of the properties of the matrix preventing the formation of crystals which could lead to an unstable printing. After deposition, it clearly appears that ILM deposition leads to the formation of a very homogeneous spots as presented Figure lc for 2,5-DHB/3AP. For each ILM, spectra were accumulated in both positive and negative mode. It clearly appears that for all ILM, similar results were obtained in positive mode (Figure 4) Concerning analysis in negative mode, all ILM present similar results expect for 2,5-DHB/DANI and 2,5-DHB/DEANI which provide spectra with weak intense peaks (Figure 5). In order to establish the efficiency of these ILM, 2,5-DHB and 2,5-DHB/3AP were deposited on the same rat brain tissue section using the symmetry axis in order to cover the same region of interest on the tissue with both matrices. Mass spectra were saved in both positive and negative mode from same region of interest and are presented Figure 6. It clearly appears that spectra obtained from the ILM and the crystalline 2,5-DHB provide ions from same classes of lipids in both negative (Figure 6c-d) and positive (Figure 6a-b) mode. However, owing to the heterogeneous crystallization presented by the 2,5-DHB, no signal was detected after irradiation in the center of the spots. On the contrary, 2,5-DHB/3AP which presents a very strong homogeneous crystallization, the distribution of analytes on the spots is very homogeneous leading to strong signal in the whole spot of 2,5-DHB/3AP which is a great advantage for MSI experiments.

To investigate the stability of ILM under vacuum, a spectrum was saved directly after the introduction in the source of a tissue section after deposition of 2,5-DHB/3AP (Figure 2c). A spectrum was again saved after lh (data not shown) in the source providing very intense signals of lipids. The target was kept in the high vacuum of the source during two days. The spectrum acquired after this time was similar than the spectrum obtain immediately after the introduction of the target in the mass spectrometer (Figure 2d).

By the high stability, the strong spot homogeneity and the ability to work in both positive and negative mode, ILM is a very good matrix suitable for MSI of lipids and other analytes. Mass Spectrometry Imaging of lipids.

In order to establish the quality of molecular images using ILM, on two whole rat brain tissue sections, 5nL of 2,5-DHB/3AP were applied on each position using the microspotter. The printing of the ILM was very stable during the deposition and due to the fact that only 5nL of solution of 2,5-DHB/3AP were necessary to obtained spots of matrices is a great advantage considering the amount a solution of 2,5-DHB or 2,6-DHAP to obtain a good crystallization. In the same conditions (20mg/mL) with 2,5-DHB or 2,6- DHAP, 40 iterations were necessary to obtain the total volume of matrix on each position. By using 2,5-DHB/3AP, only 5nL representing 10 iterations to obtain the total volume of matrix were necessary to achieved spectra of lipids with high intensity. By considering the size of the tissue section, this parameter could be a great advantage due to the fact that sample preparation of large tissue is time consuming. In our case, only 30 minutes were necessary to cover the whole rat brain tissue section with 2,5-DHB/3AP.

On both tissue sections, spots of DHB/3AP were very homogeneous as presented Figure 2c. However, it seemed that some crystals were observed outside the tissue section a. It clearly appears that this phenomenon was only observed outside the tissue and not on the tissue. The properties of DHB/3 AP on the tissue section were thus conserved allowing the acquisition of molecular images in both positive and negative mode.

The detection of two lipids presenting complementary distribution measured at m/z 753.5 and m z 788.9 in reflector positive mode corresponding respectively to the sphingomyelin (SM) {38:4} and the phosphocholine (PC) {36: 1, was performed on rat brain tissue sections and good quality images were obtained. The identification of lipids was based on the measurement of the m/z by comparison with databank established by different groups ²⁵. In positive mode, the use of ILM such as 2,5-DHB/3AP allows the detection of wide class of lipids including phosphocho lines (PC), phosphoethanolamines (PE), phosphoserines (PS) but also sphingolipids such as sphingomyelins (SM). An example of lipids detected using 2,5-DHB/3AP in positive reflector mode is presented in Table 1. Experimental mass (m/z) Theoretical mass (m z) Assignment

718.60 718.54 PE {34: 1 } [M+H]+

731.74 732.61 SM {36: 1 } [M+H]+

734.57 734.57 PC {32:0} [M+H]+

753.59 SM {36: 1 } [M+Na]+

753.59

753.59 SM {38:4} [M+H]+ 756.55 PC {34:3} [M+H]+ 756.53

756.57 PC {32:0} [M+Na]+

760.55 760.59 PC {34: 1 } [M+H]+

775.57 SM {40:7} [M+H]+

775.70

775.56 SM {38:4} [M+Na]+ 782.56 PC {34: 1 } [M+Na]+ 782.55

782.57 PC {36:4} [M+H]+

788.91 788.62 PC {36: 1 } [M+H]+

806.56 PC {36:3} [M+Na]+

806.52

806.57 PC {38:6} [M+H]+ 810.59 PC {36: 1 } [M+Na]+ 810.57

810.60 PC {38:4} [M+H]+

852.48 852.47 PS {38:6} [M-H+2Na]+

868.48 868.57 PS {38:6} [M-H+Na+K]+

Table 1: Example of lipids detected in positive reflector mode after deposition of 2,5-DHB/3AP as matrix on a rat brain tissue section. Abbreviations: phosphoethanolamine (PE), phosphocholine (PC), phosphoserine (PS), sphingomyelin (SM).

With the possibility for these ILM to work in negative mode, molecular images were performed on consecutive rat brain tissue section. The detection of two lipids presenting complementary distribution measured at m/z 806.79 and m/z 857.78 in negative reflector mode corresponding respectively to the sulfatide (ST) {36: 1 } and the phosphoinositol (PI) {36:4}, was also performed on rat brain tissue sections and good quality images were obtained. The identification of lipids was based on the measure of the m/z by comparison with databank established by different groups ⁿ' ²⁵. In this detection mode, the use of ILM such as 2,5-DHB/3AP allows the detection of different class of lipids including phospholipids such as phosphoinositols (PI) and sphingolipides such as sulfatides (ST) and gangliosides. An example of lipids detected using 2,5-DHB/3AP in negative reflector mode is presented in Table 2. Experimental mass (m/z) Theoretical mass (m/z) Assignment

806.79 806.54 ST {36:1}

834.82 834.58 ST {38:1}

850.82 850.57 ST-OH {38:1}

857.78 857.52 PI {36:4}

860.84 860.59 ST {40:2}

862.86 862.61 ST {40:1}

876.87 876.59 ST-OH {40:2}

878.86 878.59 ST-OH {40:1}

885.81 885.55 PI {38:4}

888.88 888.62 ST {42:2}

890.89 890.64 ST {42:1}

902.88 902.60 ST-OH {42:3}

904.98 90462 ST-OH {42:2}

906.89 906.63 ST-OH {42:1}

916.93 916.66 ST {44:2}

918.93 918.67 ST {44:1}

932.93 932.65 ST-OH {44:2}

934.94 934.65 ST-OH {44:1}

1544.47 1547.87 GM1 {36:1}

1572.54 1572.90 GM1 {38:1}

Table 2: Example of lipids detected in negative reflector mode after deposition of 2,5- DHB/3AP as matrix on a rat brain tissue section. Abbreviations: phosphoinositol (PI), sulfatide (ST), hydroxylated sulfatide (ST-OH).

Mass Spectrometry of drugs

In order to establish the quality of drugs detection using ILM, 2 nL of 2,5-DHB/ANI were mixed with a solution containing lOmM of Olanzapine (OLZ) in EtOH/H₂0 (7:3, v:v) and were deposited on a MALDI target. Intense peak from OLZ is detected in positive mode with a good spectral resolution as presented Figure 7.

Based on the results presented Figure 7, it clearly appears that the ILM are very efficient for drugs detection regarding to the intensity and spectral resolution of peaks from OLZ.

The repartition of Olanzapine (lOmM) in matrix 2,5-DHB in EtOH/H20 (7:3,v:v) or 2,5-DHB/ANI in EtOH/H₂0 (7:3, v:v) spot was compared. Hot spots of heterogeneous crystals are observed with the 2,5-DHB matrix whereas an homogenous signal is obtained with 2,5-DHB/ANI.

REFERENCES

I. Caprioli, . M.; Farmer, T. B.; Gile, J., Molecular imaging of biological samples: localization of peptides and proteins using MALDI-TOF MS. Anal Chem 1997, 69, (23), 4751-60.

2. Reyzer, M. L; Caprioli, R. M., MALDI-MS-based imaging of small molecules and proteins in tissues. Curr Opin Chem Biol 2007, 11, (1), 29-35.

3. Rubakhin, S. S.; Jurchen, J. C; Monroe, E. B.; Sweedler, J. V., Imaging mass spectrometry: fundamentals and applications to drug discovery. Drug Discov Today 2005, 10, (12), 823-37.

4. Hopfgartner, G.; Varesio, E.; Stoeckli, M., Matrix-assisted laser desorption/ionization mass spectrometric imaging of complete rat sections using a triple quadrupole linear ion trap. Rapid

Commun Mass Spectrom 2009, 23, (6), 733-6.

5. Chaurand, P.; Norris, J. L; Cornett, D. S.; Mobley, J. A.; Caprioli, R. M., New developments in profiling and imaging of proteins from tissue sections by MALDI mass spectrometry. J Proteome Res 2006, 5, (11), 2889-900.

6. Chaurand, P.; Sanders, M. E.; Jensen, R. A.; Caprioli, R. M., Proteomics in diagnostic pathology: profiling and imaging proteins directly in tissue sections. Am J Pathol 2004, 165, (4), 1057-68.

7. Chaurand, P.; Schwartz, S. A.; Caprioli, R. M., Imaging mass spectrometry: a new tool to investigate the spatial organization of peptides and proteins in mammalian tissue sections. Curr Opin Chem Biol 2002, 6, (5), 676-81.

8. Fournier, I.; Wisztorski, M.; Salzet, M., Tissue imaging using MALDI-MS: a new frontier of histopathology proteomics. Expert Rev Proteomics 2008, 5, (3), 413-24.

9. Franck, J.; Arafah, K.; Elayed, M.; Bonnel, D.; Vergara, D.; Jacquet, A.; Vinatier, D.;

Wisztorski, M.; Day, R.; Fournier, I.; Salzet, M., MALDI IMAGING: State of the art technology in clinical proteomics. Mol Cell Proteomics 2009.

10. Burnum, K. E.; Cornett, D. S.; Puolitaival, S. M.; Milne, S. B.; Myers, D. S.; Tranguch, S.; Brown, H. A.; Dey, S. K.; Caprioli, R. M., Spatial and temporal alterations of phospholipids determined by mass spectrometry during mouse embryo implantation. J Lipid Res 2009.

II. Dreisewerd, K.; Lemaire, R.; Pohlentz, G.; Salzet, M.; Wisztorski, M.; Berkenkamp, S.; Fournier, I., Molecular profiling of native and matrix-coated tissue slices from rat brain by infrared and ultraviolet laser desorption/ionization orthogonal time-of-flight mass spectrometry. Anal Chem 2007, 79, (6), 2463-71.

12. Woods, A. S.; Wang, H. Y.; Jackson, S. N., A snapshot of tissue glycerolipids. Curr Pharm Des 2007, 13, (32), 3344-56. 13. Sugiura, Y.; Konishi, Y.; Zaima, N.; Kajihara, S.; Nakanishi, H.; Taguchi, .; Setou, M., Visualization of the cell-selective distribution of PUFA-containing phosphatidylcholines in mouse brain by imaging mass spectrometry. J Lipid Res 2009.

14. Murphy, R. C; Hankin, J. A.; Barkley, R. M., Imaging of lipid species by MALDI mass spectrometry. J Lipid Res 2009, 50 Suppl, S317-22.

15. Han, X.; D, M. H.; McKeel, D. W., Jr.; Kelley, J.; Morris, J. C, Substantial sulfatide deficiency and ceramide elevation in very early Alzheimer's disease: potential role in disease pathogenesis. J Neurochem 2002, 82, (4), 809-18.

16. Murphy, E. J.; Schapiro, M. B.; Rapoport, S. I.; Shetty, H. U., Phospholipid composition and levels are altered in Down syndrome brain. Brain Res 2000, 867, (1-2), 9-18.

17. Petkovic, M.; Schiller, J.; Muller, M.; Benard, S.; Reichl, S.; Arnold, K.; Arnhold, J.,

Detection of individual phospholipids in lipid mixtures by matrix-assisted laser

desorption/ionization time-of-flight mass spectrometry: phosphatidylcholine prevents the detection of further species. Anal Biochem 2001, 289, (2), 202-16.

18. Schiller, J.; Suss, R.; Arnhold, J.; Fuchs, B.; Lessig, J.; Muller, M.; Petkovic, M.; Spalteholz, H.; Zschornig, O.; Arnold, K., Matrix-assisted laser desorption and ionization time-of-flight (MALDI- TOF) mass spectrometry in lipid and phospholipid research. Prog Lipid Res 2004, 43, (5), 449-88. 19. Jackson, S. N.; Wang, H. Y.; Woods, A. S., Direct profiling of lipid distribution in brain tissue using MALDI-TOFMS. Anal Chem 2005, 77, (14), 4523-7.

20. Jackson, S. N.; Wang, H. Y.; Woods, A. S., In situ structural characterization of

phosphatidylcholines in brain tissue using MALDI-MS/MS. J /A/tt Soc Mass Spectrom 2005, 16, (12), 2052-6.

21. Jackson, S. N.; Wang, H. Y.; Woods, A. S., In situ structural characterization of

glycerophospholipids and sulfatides in brain tissue using MALDI-MS/MS. J Am Soc Mass Spectrom 2007, 18, (1), 17-26.

22. Wang, H. Y.; Jackson, S. N.; Woods, A. S., Direct MALDI-MS analysis of cardiolipin from rat organs sections. J Am Soc Mass Spectrom 2007, 18, (3), 567-77.

23. Estrada, R.; Yappert, M. C, Alternative approaches for the detection of various phospholipid classes by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. J Mass Spectrom 2004, 39, (4), 412-22.

24. Jackson, S. N.; Wang, H. Y.; Woods, A. S.; Ugarov, M.; Egan, T.; Schultz, J. A., Direct tissue analysis of phospholipids in rat brain using MALDI-TOFMS and MALDI-ion mobility-TOFMS. /Am Soc Mass Spectrom 2005, 16, (2), 133-8. 25. Astigarraga, E.; Barreda-Gomez, G.; Lombardero, L; Fresnedo, O.; Castano, F.; Giralt, M. T.; Ochoa, B.; odriguez-Puertas, R.; Fernandez, J. A., Profiling and imaging of lipids on brain and liver tissue by matrix-assisted laser desorption/ ionization mass spectrometry using 2- mercaptobenzothiazole as a matrix. Anal Chem 2008, 80, (23), 9105-14.

26. Puolitaival, S. M.; Burnum, K. E.; Cornett, D. S.; Caprioli, R. M., Solvent-free matrix dry- coating for MALDI imaging of phospholipids. J Am Soc Mass Spectrom 2008, 19, (6), 882-6.

27. Hankin, J. A.; Barkley, R. M.; Murphy, R. C, Sublimation as a method of matrix application for mass spectrometric imaging. J Am Soc Mass Spectrom 2007, 18, (9), 1646-52.

28. Lemaire, R.; Tabet, J. C; Ducoroy, P.; Hendra, J. B.; Salzet, M.; Fournier, I., Solid ionic matrixes for direct tissue analysis and MALDI imaging. Anal Chem 2006, 78, (3), 809-19.

29. Djidja, M. C; Francese, S.; Loadman, P. M.; Sutton, C. W.; Scriven, P.; Claude, E.; Snel, M. F.; Franck, J.; Salzet, M.; Clench, M. R., Detergent addition to tryptic digests and ion mobility separation prior to MS/MS improves peptide yield and protein identification for in situ proteomic investigation of frozen and formalin-fixed paraffin-embedded adenocarcinoma tissue sections. Proteomics 2009, 9, (10), 2750-63.

30. Chan, K.; Lanthier, P.; Liu, X.; Sandhu, J. K.; Stanimirovic, D.; Li, J., MALDI mass

spectrometry imaging of gangliosides in mouse brain using ionic liquid matrix. Anal Chim Acta 2009, 639, (1-2), 57-61.

31. Little, D. P.; Cornish, T. J.; Odonnell, M. J.; Braun, A.; Cotter, R. J.; Koster, H., MALDI on a Chip: Analysis of Arrays of Low-Femtomole to Subfemtomole Quantities of Synthetic

Oligonucleotides and DNA Diagnostic Products Dispensed by a Piezoelectric Pipet. Anal Chem 1997, 69, 4540-4546.

32. Franck, J., Arafah, K., Barnes, A., Wisztorski, M., Salzet, M., Fournier, I., Improving tissue preparation for MALDI-MSI: Part 1, Using microspotting. Anal Chem 2009.

33. Snovida, S. I.; Chen, V. C; Perreault, H., Use of a 2,5-dihydroxybenzoic acid/aniline MALDI matrix for improved detection and on-target Derivatization of glycans: A preliminary report. Anal Chem 2006, 78, (24), 8561-8.

34. Snovida, S. I.; Perreault, H., A 2,5-dihydroxybenzoic acid/N,N-dimethylaniline matrix for the analysis of oligosaccharides by matrix-assisted laser desorption/ionization mass spectrometry. Rapid Commun Mass Spectrom 2007, 21, (22), 3711-5.

35. Snovida, S. I.; Rak-Banville, J. M.; Perreault, H., On the use of DHB/aniline and DHB/N,N- dimethylaniline matrices for improved detection of carbohydrates: automated identification of oligosaccharides and quantitative analysis of sialylated glycans by MALDI-TOF mass spectrometry. J Am Soc Mass Spectrom 2008, 19, (8), 1138-46. 36. Tholey, A., Ionic liquid matrices with phosphoric acid as matrix additive for the facilitated analysis of phosphopeptides by matrix-assisted laser desorption/ionization mass spectrometry. Rapid Commun Mass Spectrom 2006, 20, (11), 1761-8.

37. Palmblad, M.; Cramer, ., Liquid matrix deposition on conductive hydrophobic surfaces for tuning and quantitation in UV-MALDI mass spectrometry. J Am Soc Mass Spectrom 2007, 18,

(4), 693-7.

Claims

1. Method for detection and/or quantification of an analyte in a biological tissue section comprising the following steps:

a. Depositing a liquid ionic matrix onto the biological tissue section wherein the liquid ionic matrix comprises 2,5-DHB and an organic base,

b. Analysing the biological tissue by Matrix Assisted Laser Desorption Mass spectrometry Imaging.

2. Method for detection and/or quantification of an analyte in a biological tissue section according to claim 1 wherein the liquid ionic matrix is selected from 2,5-DHB/3AP, 2,5-DHB/ANI, 2,5-DHB/Pyr, 2,5-DHB/DANI and/or 2,5- DHB/DEANI.

3. Method for detection and/or quantification of an analyte in a biological tissue section according to anyone of claims 1-2 wherein the liquid ionic matrix is deposited onto the biological tissue section by micro-spotting.

Method for detection and/or quantification of an analyte in a biological tissue section according to anyone of claims 1-3 wherein at least one micro-spot of between 5 and 15 nL of ionic liquid matrix is deposited onto the biological tissue section.

Method for detection and/or quantification of an analyte in a biological tissue according to anyone of claims 1-4 wherein a defined pattern of micro-spots of ionic liquid matrix is deposited onto the biological tissue section.

Method for detection and/or quantification of an analyte in a biological tissue section according to anyone of claims 1-5 wherein the analyte is a lipid.

7. Method for detection and/or quantification of an analyte in a biological tissue section according to anyone of claims 1-6 wherein the analyte is selected from phospoethanolamines (PE), phosphocho lines (PC), phosphoserines (PS), sphingomyelins, phosphoinositols (PI), sulfatides (ST), hydroxylated sulfatides (ST-OH).

8. Method for detection and/or quantification of an analyte in a biological tissue section according to anyone of claims 1-5 wherein the analyte is a small molecule having a size comprised between 23 and 2000 M/z.

9. Method for detection and/or quantification of an analyte in a biological tissue section according to anyone of claims 1-8 wherein the biological tissue section is analysed by Matrix Assisted Laser Desorption Mass spectrometry Imaging in both positive and negative reflector mode.

10. Method for detection and/or quantification of an analyte in a biological tissue section according to anyone of claims 1-9 wherein the analyte is both detected and quantified in the biological tissue section.

11. Composition comprising between 5 and 30 mg/mL 2,5-DHB in a solvent and an organic base selected from 3AP, ANI, Pyr, DANI and/or DEANI.

12. Composition according to claim 11 wherein the ratio organic base/2,5-DHB is comprised between 0,5 and 2.

13. Composition according to anyone of claims 11-12 wherein the solvent

comprises methanol, ethanol and TFA.

14. Use of a composition according to anyone of claims 11-13 for detection and/or quantification of an analyte in a biological tissue section by Matrix Assisted Laser Desorption Mass spectrometry Imaging.

15. Use of a composition for detection and/or quantification of an analyte in a biological tissue section by Matrix Assisted Laser Desorption Mass

spectrometry Imaging according to claim 14 wherein the analyte is a lipid.