Title: On-tissue Direct Monitoring of Global Hydrogen/Deuterium Exchange by MALDI Mass Spectrometry: Tissue Deuterium Exchange Mass Spectrometry (TDXMS)
Abstract: Hydrogen/deuterium exchange mass spectrometric (H/DXMS) methods for protein structural analysis are conventionally performed in solution. We present Tissue Deuterium Exchange Mass Spectrometry (TDXMS), a method to directly monitor deuterium uptake on tissue, as a means to better approximate the deuterium exchange behavior of proteins in their native microenvironment. Using this method, a difference in deuterium uptake behavior was observed when the same proteins were monitored in solution and on tissue. The higher maximum deuterium uptake at equilibrium for all proteins analyzed in solution suggests a more open conformation in the absence of interacting partners normally observed on tissue. We also demonstrate a difference in the deuterium uptake behavior of a few proteins across different morphological regions of the same tissue section. Modifications of the total number of hydrogens exchanged, as well as the kinetics of exchange, were both observed. These results provide information on the implication of protein interactions with partners as well as on the conformational changes related to these interactions, and illustrate the importance of examining protein deuterium exchange behavior in the presence of its specific microenvironment directly at the level of tissues. Hydrogen/deuterium exchange mass spectrometric (H/DXMS) methods for protein structural analysis are conventionally performed in solution. We present Tissue Deuterium Exchange Mass Spectrometry (TDXMS), a method to directly monitor deuterium uptake on tissue, as a means to better approximate the deuterium exchange behavior of proteins in their native microenvironment. Using this method, a difference in deuterium uptake behavior was observed when the same proteins were monitored in solution and on tissue. The higher maximum deuterium uptake at equilibrium for all proteins analyzed in solution suggests a more open conformation in the absence of interacting partners normally observed on tissue. We also demonstrate a difference in the deuterium uptake behavior of a few proteins across different morphological regions of the same tissue section. Modifications of the total number of hydrogens exchanged, as well as the kinetics of exchange, were both observed. These results provide information on the implication of protein interactions with partners as well as on the conformational changes related to these interactions, and illustrate the importance of examining protein deuterium exchange behavior in the presence of its specific microenvironment directly at the level of tissues. Hydrogen/Deuterium Exchange Mass Spectrometry is a robust technique that can be used to study structural changes of proteins. It has been demonstrated, for example, in the study of protein folding dynamics (1.Smirnovas V. Kim JI. Lu X. Atarashi R. Caughey B. Surewicz WK. Distinct structures of scrapie prion protein (PrPSc)-seeded versus spontaneous recombinant prion protein fibrils revealed by hydrogen/deuterium exchange.J. Biol. Chem. 2009; 284: 24233-24241Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), and protein-protein (such as antigen-antibody interactions (2.Tu T. Drăguşanu M. Petre BA. Rempel DL. Przybylski M. Gross ML. Protein-peptide affinity determination using an h/d exchange dilution strategy: application to antigen-antibody interactions.J. Am. Soc. Mass Spectrom. 2010; 21: 1660-1667Crossref PubMed Scopus (22) Google Scholar), allosteric binding (3.Seckler J.M. Barkley M.D. Wintrode P.L. Allosteric suppression of HIV-1 reverse transcriptase structural dynamics upon inhibitor binding.Biophys. J. 2011; 100: 144-153Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), aggregation (4.Kraus M. Bienert M. Krause E. Hydrogen exchange studies on Alzheimer's amyloid-beta peptides by mass spectrometry using matrix-assisted laser desorption/ionization and electrospray ionization.Rapid Commun. Mass Spectrom. 2003; 17: 222-228Crossref PubMed Scopus (28) Google Scholar), etc.) and protein-ligand interactions in solution (5.Zhang J. Chalmers MJ. Stayrook KR. Burris LL. Garcia-Ordonez RD. Pascal BD. Burris TP. Dodge JA. Griffin PR. Hydrogen/deuterium exchange reveals distinct agonist/partial agonist receptor dynamics within vitamin D receptor/retinoid X receptor heterodimer.Structure. 2010; 18: 1332-1341Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). This can be done by analyzing global changes observed by subjecting the protein of interest to deuterium exchange and analyzing the amount of deuterium incorporation directly (6.Wales TE Engen JR Hydrogen exchange mass spectrometry for the analysis of protein dynamics.Mass Spectrom. Rev. 2006; 25: 158-170Crossref PubMed Scopus (678) Google Scholar). It can also be used to determine the specific regions that are affected by conformational change, by digesting the protein using pepsin, and analyzing the subsequent peptides generated after doing a rapid chromatographic separation ensuring that the extent of back exchange will be minimal (6.Wales TE Engen JR Hydrogen exchange mass spectrometry for the analysis of protein dynamics.Mass Spectrom. Rev. 2006; 25: 158-170Crossref PubMed Scopus (678) Google Scholar). Several methods exist for the study of protein conformation by deuterium exchange in addition to mass spectrometry, such as Fourier Transform Infrared Spectroscopy (FTIR), Circular Dichroism (CD), and Nuclear Magnetic Resonance (NMR). Methods such as FTIR (7.Sinha S. Li Y. Williams T.D. Topp E.M. Protein conformation in amorphous solids by FTIR and by hydrogen/deuterium exchange with mass spectrometry.Biophys. J. 2008; 95: 5951-5961Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 8.Dong A. Matsuura J. Allison SD. Chrisman E. Manning MC. Carpenter JF. Infrared and circular dichroism spectroscopic characterization of structural differences between beta-lactoglobulin A and B.Biochemistry. 1996; 35: 1450-1457Crossref PubMed Scopus (153) Google Scholar) and CD (8.Dong A. Matsuura J. Allison SD. Chrisman E. Manning MC. Carpenter JF. Infrared and circular dichroism spectroscopic characterization of structural differences between beta-lactoglobulin A and B.Biochemistry. 1996; 35: 1450-1457Crossref PubMed Scopus (153) Google Scholar) are normally restricted to global conformational change studies. Far ultraviolet (UV) CD can be used to study secondary structural elements whereas near UV CD can be used to study tertiary structure focusing mainly on aromatic amino acid residues. Positional specificity down to the amino acid level can be achieved using NMR; it is limited however to the analysis of low mass proteins typically below 30 kDa ((9.Kuwata K. Matumoto T. Cheng H. Nagayama K. James TL. Roder H. NMR-detected hydrogen exchange and molecular dynamics simulations provide structural insight into fibril formation of prion protein fragment 106–126.Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 14790-14795Crossref PubMed Scopus (121) Google Scholar), for example); beyond this the complexity of the analysis of 2D NMR correlation increases. Protein structure is mainly studied by x-ray crystallography, and like NMR, it provides unequivocal information down to single amino acid positional specificity. A limitation of this technique though is the need to crystallize the protein, thus, not only restricting the analysis to its stable, crystal conformation, but also requiring that the protein to be analyzed is in its purest form (10.Kaltashov I.A. Bobst C.E. Abzalimov R.R. Berkowitz S.A. Houde D. Conformation and dynamics of biopharmaceuticals: transition of mass spectrometry-based tools from academe to industry.J. Am. Soc. Mass Spectrom. 2010; 21: 323-337Crossref PubMed Scopus (83) Google Scholar). In addition, it is difficult to study proteins with partial amorphous structures using this technique because they are difficult to crystallize. Compared with the last two techniques mentioned, H/DXMS currently offers only limited positional specificity defined by the extent which the proteolytic enzyme used cleaves the protein, but it can be advantageous in other respects. For example, it only requires picomoles of protein and does not require the protein to be of high purity because of the high sensitivity offered by recent advances in the MS instrumentation (11.Houde D. Berkowitz S.A. Engen J.R. The utility of hydrogen/deuterium exchange mass spectrometry in biopharmaceutical comparability studies.J. Pharm. Sci. 2011; 100: 2071-2086Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). Also, the analysis has been demonstrated for large macromolecular complexes having masses above 180 kDa, such as the brome mosaic virus (12.Wang L. Lane L.C. Smith D.L. Detecting structural changes in viral capsids by hydrogen exchange and mass spectrometry.Protein Sci. 2001; 10: 1234-1243Crossref PubMed Scopus (70) Google Scholar). Despite its successful application, deuterium exchange experiments are still restricted to studies in solution. Indeed, there has been a gradual shift toward performing protein dynamics studies when interacting with their stable ligands and other small or polymeric biomolecules in an attempt to mimic the behavior of these proteins in vivo. By taking the protein dynamics in vitro to be identical in vivo, however, these studies suffer the risk of underestimating the extent of interactions that might be present in the protein microenvironment which varies depending on its localization (13.Kaltashov I.A. Bobst C.E. Abzalimov R.R. H/D exchange and mass spectrometry in the studies of protein conformation and dynamics: is there a need for a top-down approach?.Anal. Chem. 2009; 81: 7892-7899Crossref PubMed Scopus (113) Google Scholar). Recent evidence shows that when deuterium exchange is performed in vivo, the thermodynamic stability of the protein studied increased in response to a hyperosmotic change in its environment, compared when the protein is analyzed in vitro (14.Ghaemmaghami S. Oas T.G. Quantitative protein stability measurement in vivo.Nat. Struct. Biol. 2001; 8: 879-882Crossref PubMed Scopus (157) Google Scholar). In this work, the protein was overexpressed in E. coli cells, which were then exposed to deuterated media to enable the incorporation of deuterium inside the cells via passive diffusion. The idea of studying proteins in vivo using deuterium exchange has been described as early as 1930 (15.Bonhoeffer K.F. Schweres wasser.Angew Chem. 1933; 46: 776-779Crossref Google Scholar), but until recently only the aforementioned publication has described the application of MS to study this. The major drawback of the aforementioned study was its limited application to single cell organisms where the protein has been overexpressed. Overexpression of the protein of interest allowed its detection and enabled deuterium exchange experiments to be performed on it. This means though that the protein is not anymore in its native environment where it may have different interacting partners which may not be reflected anymore in the case of the single cell organism. Furthermore, in order to express the protein on the host cell, modifications such as truncation of several amino acid sequences might be necessary. If we want to study protein dynamics in its native state, a wise approach would be to examine them in the tissues where they are originally expressed. This is however difficult to do in practice, because of limitations imposed by the deuterium exchange method itself. In this study, we applied the deuterium exchange approach to study proteins on tissue to determine if the deuterium uptake is affected in the tissue by comparison to the solution and if localization on the tissue matrix has also significant effects. Direct MS measurements on tissue are advantageous in that the analysis is rapid, information can be obtained on hundreds of molecular signatures at the same time, and direct measurements of proteins/peptides are performed on tissue. In this report, we show that the effect of the tissue environment has varying effects on the deuterium uptake depending on the examined protein. HPLC grade water, trifluoroacetic acid (TFA), acetonitrile (ACN) and ammonium acetate (NH4OAc) were obtained from Biosolve BV (Dieuze, France). Sinapinic acid (SA, puriss) and deuterated water (D2O) enriched up to 99.9% with deuterium were purchased from Sigma-Aldrich (St. Quentin-Fallavier, France). Bovine ubiquitin and chick lysozyme C protein standards were purchased from Promega (Charbonnières-les-Bains, France) and were used without further purification. The deuterium exchange procedure used to analyze protein standards was performed as follows. Lysozyme C and Ubiquitin standards were prepared in 100 μl of 50 mm NH4OAc in H2O (pH 7.6) at a concentration of 1.5 mg/ml each. The mixture was allowed to equilibrate for at least an hour before working solutions were obtained. To realize deuterium exchange, 3 μl working solution was diluted in 30 μl 50 mm NH4OAc prepared in D2O. 1 μl of the solution was taken for every time point and diluted to 10 μl with a quenching buffer (2% TFA in H2O, pH = 2.2). The solution was immediately frozen in liquid nitrogen until analysis. To examine the amount of deuterium uptake, the frozen samples were partly melted and 0.5 μl was spotted on a MALDI target. To this, 0.5 μl ice-cold matrix solution composed of 20 mg/ml SA suspended in 500 μl 2% TFA in H2O + 500 μl ACN/0.1% TFA in H2O (7:3, v/v) (quenching matrix solution) was added. The spots were dried for at least 5 min under vacuum in a cold room (4 °C) and analyzed immediately. A 12-μm coronal section of fresh frozen rat brain was dispersed in 10 μl 50 mm NH4OAc in H2O by vortex mixing after placing the tissue section in a micro-tube. The mixture was then centrifuged for 10 min at 10,000 × g and the supernatant was collected and dried in a speedvac. The dried extracts were reconstituted with 10 μl 50 mm NH4OAc in H2O and allowed to equilibrate for at least an hour before solutions were taken for deuterium uptake experiments. Three microliters of the extract was diluted in 30 μl 50 mm NH4OAc made up in D2O to achieve exchange. A 0.5 μl sample was taken for each time point and spotted on a MALDI target, to which 0.5 μl of the quenching matrix solution was added. The spots were dried under vacuum for at least 5 min and analyzed immediately. On-tissue deuterium exchange analysis was done as follows. Serial 12-μm coronal rat brain sections were obtained using a cryostat and mounted on ITO-covered glass slides, at one section per slide. The sections were dried under vacuum for 15 min. A 10 μl sample of 50 mm NH4OAc was then deposited throughout the entire section, and each slide was placed inside a Petri dish. The Petri dish contained paper towels wetted with 50 mm NH4OAc solution to prevent drying of the deposited liquid. The sections were allowed to equilibrate for at least 1 h before use, after which the excess liquid was withdrawn and replaced with 15 μl of 50 mm NH4OAc made up in D2O to achieve deuterium exchange. The slides were replaced in a Petri dish also containing paper towels wetted with 50 mm NH4OAc but this time made up in D2O. One section was prepared for each time point. During each time point, the exchange solution of the corresponding section was pipetted off and 10 μl quenching matrix solution was deposited throughout the entire section. The section was then dried under vacuum for at least 5 min and analyzed immediately. A control section was prepared following the same procedure but with H2O replacing D2O in all solutions. In experiments monitoring the deuterium exchange of the proteins across different regions, sagittal sections of the rat brain were used. The same protocol was applied except that only 1 μl of the exchange solution and quenching matrix solution were used per region, allowing for local H/D exchanges to occur and reducing the drying time to not more than 2 min. Mass spectrometric analysis was performed using a MALDI UltraFlex II time-of-flight (TOF) instrument equipped with a Smartbeam laser (355 nm emission wavelength) having a maximum repetition rate of 200 Hz and controlled by a FlexControl 3.0 (Build 158) software from Bruker Daltonics, GmbH (Bremen, Germany). The full MS spectra were acquired in positive linear mode with delayed extraction within the 3000–20,000 m/z range by accumulating 5000 laser shots at a repetition rate of 200 Hz. The laser energy was fixed to be just slightly above the threshold energy for ion detection. Medium deflection of the low mass ions below m/z 3000 was used to enhance the detection of proteins. The MALDI target was always calibrated prior to analysis using Protein Calibration Standard 1 (Bruker Daltonics, GmbH), which contains a mixture of six proteins covering a mass range of m/z 5500–17,000. Mass values expressed in the text correspond to the centroid of the m/z values of the [M + H]+ ions. The MALDI-TOF raw data of the H/D exchanges have been deposited at the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD004630. Proteins were extracted from a consecutive 12-μm rat brain tissue section. One microliter of the protein extract was suspended in either 10 or 30 μl 5:95 ACN/0.1% FA in H2O and 4 μl of these samples were injected in a 200 μm × 50 cm ProSwift RP-4H monolithic capillary column (Thermo Fisher Scientific, Bremen, Germany). Proteins were eluted using a linear gradient from 3% to 40% ACN for 60 min, then to 95% ACN for 10 min, followed by a wash out until 90 min, at a flow rate of 1.4 μl/min. Spectra were acquired in data-dependent mode using a an LTQ Orbitrap XL mass spectrometer and subjecting the top 2 most intense peaks for fragmentation. The full MS scans were acquired at 100,000 resolution for m/z 400 and acquiring 2 FTMS microscans at a maximum ion injection time of 1000 ms. On the other hand, the MS/MS scans were acquired at 60,000 resolution for m/z 400 and acquiring four microscans at a maximum injection of 500 ms. Higher-energy collision dissociation (HCD) activation was used with the activation time set at 1s. The isolation width was set at ±15 ppm and the default charge state at 10. The normalized collision energy (NCE) was fixed at 30.0 V. Dynamic exclusion was enabled and the exclusion mass width was set to ±9 ppm by mass. The repeat count was set to 1 and repeat duration to 240 s. The exclusion list size was set to 500 and the exclusion duration to 180 s. +1, +2 and +3 charge states were rejected. For top-down analysis, the data were analyzed with ProSightPC 3.0 (Thermo Fisher Scientific) and Proteome Discoverer 2.1 (Thermo Fisher Scientific) utilizing the ProSightPD 1.0 node. Spectra were then searched using a three-tiered search tree. The first search was an Absolute Mass search with MS1 tolerance of 100 Da, MS2 tolerance of 10 ppm, against a Rattus norvegicus UniProt database containing 35,953 canonical and isoform sequences accessed on July 8, 2016, or the ProSightPD 1.0 database available at their website (ftp://prosightpc.northwestern.edu/). The second search was a ProSight Biomarker search with MS1 tolerance of 10 ppm, MS2 tolerance of 10 ppm, against the same databases. Finally, a second Absolute Mass search was performed with MS1 tolerance of 1000 Da, MS2 tolerance of 10 ppm, using Delta M mode, against the same databases. Protein assignments can be found in supplemental Data S1. The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the same data set identifier (PXD004630). The *.xml Search files for 20160719_TopDown2 correspond to searches done using the UniProt database, whereas those for 20160719_topDown correspond to searches performed using the ProSightPD database. hydrogen/deuterium trifluoroacetic acid hydrogen/deuterium exchange mass spectrometry Fourier Transform infrared Spectroscopy Circular Dichroism Nuclear Magnetic Resonance ammonium acetate deuterium oxide sinapinic acid acetonitrile matrix-assisted laser desorption/ionization. Optimization of the deuterium exchange protocol in solution for analysis using MALDI-TOF MS was first performed using standard proteins. The most crucial aspects of H/D exchange optimization from tissues are finding solvent systems that both preserve protein folding and remain compatible with MS analysis, and control of the back exchange process that will rapidly occur once the equilibrium of exchange has been achieved. Here we used a protein mix composed of bovine ubiquitin and chicken lysozyme which was first equilibrated in a buffer containing a kosmotrophic salt such as NH4OAc for at least an hour to ensure that the exchange was performed on the nondenatured conformation of the proteins. NH4OAc is suitable for MALDI-TOF MS because it is volatile and contributes minimal interference during MS experiments (16.Asara J.M. Allison J. Enhanced detection of oligonucleotides in UV MALDI MS using the tetraamine spermine as a matrix additive.Anal. Chem. 1999; 71: 2866-2870Crossref PubMed Scopus (56) Google Scholar). pH and temperature play significant roles in the extent of back exchange that ensues immediately after deuterium uptake has taken place. To minimize its effect, the exchange solution must be brought to pH = 2.4 and the temperature to 0 °C, and the samples immediately analyzed. In our protocol, we found that dilution of a saturated matrix solution with 2% TFA is sufficient to decrease the pH to 2.2 without significantly affecting the protein signals during MS acquisition or further increasing the drying time of the sample spots on the MALDI target (less than 10 min). To decrease the temperature, the acidified matrix solution was chilled in ice prior to spotting on the target; the matrix solution is sonicated and vortexed regularly to ensure that sufficient matrix is dissolved in solution before use. We restricted this study to global analysis because we want to establish first if there will be significant effects when the protein is analyzed in solution and on tissue, suggesting a difference in protein conformation because of differences in interacting partners. A MALDI source is ideal for this purpose because the predominant ions observed within our specified mass range are [M+H]+ protonated species, thus simplifying the discrimination of peaks. Fig. 1 shows the deuterium exchange curves for lysozyme C and ubiquitin in solution. In both instances, the average deuterium uptake rapidly increases in the initial stages of the exchange, then gradually increases and tapers after 28 h of D2O incubation, although the uptake still continues even after extended periods of incubation (8 days). This gradual increase in deuterium uptake is typical for native proteins in solution (17.Rutkowska-Wlodarczyk I. Kierdaszuk B. Wlodarczyk J. Analysis of proton exchange kinetics with time-dependent exchange rate.Biochim. Biophys. Acta. 2010; 1804: 891-898Crossref PubMed Scopus (2) Google Scholar), and is in contrast to the rapid deuterium uptake of denatured proteins where the maximum deuterium incorporation is easily obtained in a matter of minutes (18.Chung E.W. Nettleton EJ. Morgan CJ. Gross M. Miranker A. Radford SE. Dobson CM. Robinson CV. Hydrogen exchange properties of proteins in native and denatured states monitored by mass spectrometry and NMR.Protein Sci. 1997; 6: 1316-1324Crossref PubMed Scopus (93) Google Scholar). The latter is confirmed in our initial attempts with lysozyme C that was dissolved in 0.1% TFA, spotted and dried on a MALDI target, and reconstituted with D2O, where 126 deuteriums were already incorporated within the first 60s of exchange even when the disulfide bridges were not cleaved (data not shown). This is well in line with the proteins conformation data. Indeed, Ubiquitin is a globular protein containing 144 labile hydrogens, 72 in the amide backbone and 72 in the amino acid side chains (19.Katta V. Chait B.T. Hydrogen/Deuterium Exchange Electrospray Ionization Mass Spectrometry: A Method for Probing Protein Conformational Changes in Solution.J. Am. Chem. Soc. 1993; 115: 6317-6321Crossref Scopus (261) Google Scholar). The tightly folded protein contains an α helix at residues Ile23-Glu34, and two β strands adjacent to each other at residues Met1-Thr7 and Thr12-Glu16 comprising a mixed β sheet. The α helix backbone amide hydrogens are virtually nonexchangeable because of their strong interaction with adjacent carbonyl oxygens, whereas those of the β strands are slow exchanging (20.Yu H.D. Ahn S. Kim B. Protein structural characterization by hydrogen/deuterium exchange mass spectrometry with top-down electron capture dissociation.Bull. Korean Chem. Soc. 2013; 34: 1401-1446Crossref Scopus (3) Google Scholar). These structures, together with uncorrected back exchange (21.Mandell J.G. Falick A.M. Komives E.A. Measurement of amide hydrogen exchange by MALDI-TOF mass spectrometry.Anal. Chem. 1998; 70: 3987-3995Crossref PubMed Scopus (185) Google Scholar), limit the maximum deuterium incorporation observed to 53%. A similar and even more pronounced effect is observed in the deuterium uptake of lysozyme C; which is also a globular protein containing a well-protected core stabilized by four disulfide bridges. This region is comprised of 30 well-protected backbone amide hydrogens that do not exchange even after 14 days (17.Rutkowska-Wlodarczyk I. Kierdaszuk B. Wlodarczyk J. Analysis of proton exchange kinetics with time-dependent exchange rate.Biochim. Biophys. Acta. 2010; 1804: 891-898Crossref PubMed Scopus (2) Google Scholar, 18.Chung E.W. Nettleton EJ. Morgan CJ. Gross M. Miranker A. Radford SE. Dobson CM. Robinson CV. Hydrogen exchange properties of proteins in native and denatured states monitored by mass spectrometry and NMR.Protein Sci. 1997; 6: 1316-1324Crossref PubMed Scopus (93) Google Scholar). The deuterium exchange protocol was then adapted to examine the deuterium uptake of proteins on tissue sections. For on-tissue experiments, the choice, order and combination of D2O, quench acid, and matrix are crucial parameters to be able to observe deuterium exchange directly on tissue, while avoiding protein denaturation and minimizing back exchange processes. These parameters thus need to be carefully studied and optimized. Fig. 2 presents the designed strategy to perform and measure the deuterium uptake of proteins on tissue sections. To prevent denaturation of the proteins on tissue, the sections were immediately incubated in NH4OAc buffer for at least 1h after sectioning and drying under vacuum. After incubation of the tissue sections in NH4OAc, the buffer was aspirated and the sections were transferred to a medium saturated with the deuterated buffer prior to addition of the exchange solution onto the tissue section. The deuterated buffer in the medium prevents drying of the exchange solution and the possible gas phase exchange that occurs between the exchange solution and the environment. When the Petri dish was sealed with parafilm, it was observed that the exchange solution can be maintained for extended periods (6 days in these experiments) without drying. This workflow is designed to monitor the proteins that remain within their local environment during the H/D exchange, avoiding de facto, the contribution of the proteins undergoing diffusion or extraction (here the lower mass soluble proteins) during the conditioning or exchanging step. Indeed, the exchange solution is removed just before adding the matrix solution that is used to quench the reaction. Nonetheless, to check this aspect we analyzed the NH4OAc buffer used for on-tissue conditioning and compared it with that of the tissue after removing the conditioning solution (supplemental Fig. S2). The MALDI-TOF MS spectrum of the conditioning medium reveals much fewer signals thanthe on-tissue one. The observed signals are common with the tissue and correspond to the highly abundant proteins that are of the lowest mass and highest solubility. Some of these show very high intensities compared with the tissue because of the lower complexity of the studied system. This demonstrates that only a small portion of a few abundant and soluble proteins diffuse into the buffer solution and that the largest majority of the proteins remain within their local environment. In order to demonstrate the importance of the tissue environment on the protein conformation, we need to compare the H/D exchange kinetics for the same proteins both in solution and in the tissue context. Extraction of intact proteins from a consecutive tissue section using the NH4OAc buffer allowed us to obtain proteins whose rate of deuterium uptake can be monitored both in solution and on the tissue section. Results show that signals can be detected in linear positive mode from an extract obtained from an entire tissue section (Fig. 3A, top). Likewise, the full MS spectrum acquired from the tissue section incubated in NH4OAc buffer also showed abundant signals (Fig. 3A, bottom), even when no lipid removal steps were incorporated into the protocol to enhance the detection of intact proteins (22.Lemaire R. Wisztorski M. Desmons A. Tabet JC. Day R. Salzet M. Fournier I. MALDI-MS direct tissue analysis of proteins: Improving signal sensitivity using organic treatments.Anal. Chem. 2006; 78: 7145-7153Crossref PubMed Scopus (152) Google Scholar). More importantly, several of the signals from the full MS spectrum of the extracted proteins in solution can be matched with those observed from the direct tissue analysis, as shown in Fig. 3 and supplemental Data S1 allowing for the comparison of the deuterium uptake in solution versus in the tissue. In order to identify the proteins common to the tissue extracts and in situ on the tissue, proteins were extracted from a consecutive tissue section of same thickness and submitted to Top-Down analysis. Extra