Formalin-Fixed Paraffin-Embedded (FFPE) - ACS Publications

Formalin-Fixed Paraffin-Embedded (FFPE) Proteome Analysis Using Gel-Free and Gel-Based Proteomics Omid Azimzadeh,*,† Zarko Barjaktarovic,† Michaela Aubele,‡ Julia Calzada-Wack,‡ Hakan Sarioglu,§ Michael J. Atkinson,| and Soile Tapio*,† Helmholtz Zentrum Mu ¨ nchen, German Research Center for Environmental Health, Institute of Radiation Biology, 85764 Neuherberg, Germany, Helmholtz Zentrum Mu ¨ nchen, German Research Center for Environmental Health, Institute of Pathology, 85764 Neuherberg, Germany, Helmholtz Zentrum Mu ¨ nchen, German Research Center for Environmental Health, Department of Protein Science, Proteomics Core Facility, Neuherberg, Germany, and Department of Radiation Oncology, Klinikum Rechts der Isar, Technische Universita¨t Mu ¨ nchen, Munich, Germany Received May 7, 2010

Formalin-fixed paraffin-embedded (FFPE) tissue has recently gained interest as an alternative to fresh/ frozen tissue for retrospective protein biomarker discovery. However, during the fixation process, proteins undergo degradation and cross-linking, making conventional protein analysis technologies problematic. In this study, we have compared several extraction and separation methods for the analysis of proteins in FFPE tissues. Incubation of tissue sections at high temperature with a novel extraction buffer (20 mM Tris-HCl, pH 8.8, 2% SDS, 1% β-octylglucoside, 200 mM DTT, 200 mM glycine, and a mixture of protease inhibitors) resulted in improved protein recovery. Protein separation by 1-DE followed by LC-ESI MS/MS analysis was the most effective approach to identify proteins, based on the number of peptides reliably identified. Interestingly, a number of peptides were identified in regions of the 1DE not corresponding to their native molecular weights. This is an indication of the formation of protein-protein complexes by cross-linking, and of protein fragmentation due to prolonged sample storage. This study will facilitate the development of future proteomic analysis of FFPE tissue and provide a tool for the validation in archival samples of biomarkers of exposure, prognosis and disease. Keywords: formalin-fixed paraffin-embedded (FFPE) • gel-based • gel-free • proteomics • protein extraction • peptide modification • cross-linking

1. Introduction Formalin-fixed paraffin-embedded (FFPE) tissues are routinely prepared from biopsies and at autopsy, and are held in vast quantities for many years. These tissues are proving a valuable source for the retrospective analysis of DNA and RNA, where technologies exist to prepare usable samples from histologically defined tissues. Recently, FFPE tissues have also been considered as an alternative to fresh/frozen tissue for protein biomarker discovery,1 especially when in many cases the large clinical archives represent the only source of biomaterial available. However, the harsh and irreversible fixation conditions and the prolonged storage times have made retrospective biological studies on a proteome level a difficult task. Simultaneously, the analysis of the FFPE proteome has been hampered by the chemical content of the extraction buffers needed to effectively extract proteins (e.g., detergents, reductants, denaturants, and salts). These conditions render the * To whom correspondence should be addressed. E-mail: omid.azimzadeh@ helmholtz-muenchen.de; [email protected]. † Institute of Radiation Biology. ‡ Institute of Pathology. § Department of Protein Science, Proteomics Core Facility. | Technische Universita¨t Mu ¨ nchen.

4710 Journal of Proteome Research 2010, 9, 4710–4720 Published on Web 07/06/2010

protein extracts incompatible with the subsequent steps required for a proteomics approach. Although several alternatives have been developed to optimize protein extraction and separation using FFPE tissue,2-5,8 the qualitative and quantitative proteome analysis remains a challenge. In early studies, the heat-induced antigen retrieval procedure was used to extract proteins from FFPE tissue sections before separation by SDS-PAGE.3-5 This method was used effectively for the detection of full-length proteins on Western blot,6 reverse phase protein microarray analysis,7 and post-translational modification studies.8 Unfortunately, this procedure fails to provide sufficient material that is compatible with conventional protein analysis methodologies, such as classical 2D gel electrophoresis.5,9,10 Several studies have been conducted to overcome the adverse effects of cross-linking on the quality and quantity of the protein extracted from FFPE. To improve extraction protocols Fowler et al. developed a tissue surrogate as a model system for FFPE samples to analyze the effect of the degree of cross-linking on the quality of the extraction The authors showed that exposure of the FFPE surrogate to the high temperatures (90 °C) in acidic (pH 5) buffers results in efficient protein extraction.11 Using extraction buffers with various pH 10.1021/pr1004168

 2010 American Chemical Society

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FFPE Proteome Analysis Using Gel-Free and Gel-Based Proteomics values, Shi et al. analyzed the FFPE proteomes of human breast tissue by mass spectrometry, combined with capillary isoelectric focusing (CIEF) and reversed-phase liquid chromatography (RPLC).12 Recently, Nirmalan et al. identified comparable but not identical protein profiles of FFPE and fresh/frozen tissue using surface-enhanced laser desorption ionization-time of flight mass spectroscopy (SELDI-TOF) analysis after boiling samples in Laemmli buffer.13 Only a few studies so far have reported the application of a classical 2D electrophoresis approach for the analysis of the FFPE tissue; these reports mainly confirming the unsuitability of this material for such an analysis.5,9,10 Ono et al. performed a quantitative study of FFPE versus fresh/frozen tissue using two-dimensional difference in gel electrophoresis (2D-DIGE). The low quality of the stained 2D gel enabled the identification of only a few deregulated protein spots.10 Recently, Addis et al. used sheep skeletal muscle and liver as standard tissue models to exclude the effect of the variability between FFPE samples, arising mainly from different preparation procedures but also from other factors such as storage age. Using this standard FFPE model, the authors were able to improve the 2D electrophoretic pattern of FFPE extracted proteins.14 Gel-free proteomic studies using label-free approach or alternative labeling strategies such as iTRAQ have been more successful than the gel-based methods in the number of identified proteins in FFPE material.15,16 Several studies have used shotgun proteomics strategy and liquid chromatography after a direct in-tissue digestion. In these studies, the compatibility of different extraction buffers with trypsin digestion was examined.17-21 Sprung et al. compared the protein profiles of the FFPE and fresh/frozen colon adenoma tissue by multidimensional LC-MS/MS after isoelectric focusing of the peptides.21 Using commercial protein preparation buffer (LiquidTissue) and stable isotopic labeling, Hood et al. performed a quantitative proteomic analysis on FFPE prostate tissue after nanoreverse-phase LC (nano-RPLC) tandem mass spectrometry.22 Scicchitano et al. applied the same buffer to compare the protein profile of the FFPE and fresh/frozen rat liver. The identification of a greater number of proteins in FFPE samples compared to fresh/frozen samples led the authors to conclude that the number of identified unique proteins in the FFPE samples was not dramatically affected by the formalin-fixation and paraffin-embedding procedure.20 These data were consistent with a shotgun proteomic study done on formalin-fixed mouse liver tissue, the results indicating that fixation does not necessarily compromise the proteome coverage.18 Sprung et al. used a detergent-free in-tissue digestion approach for extraction of peptides form colon adenoma FFPE and fresh/frozen samples and separated the generated peptides by isoelectric focusing before identification by LC MS-MS. The results showed a significant overlap in proteins identified between the two samples.21 Using multidimensional separation platform including capillary isoelectric focusing (CIEF)/nanoRPLC, Guo et al. separated the tryptic peptides from microdissected FFPE glioblastoma.23 Negishi et al. described a labelfree quantitative proteomics method using the 2-dimensional image-converted analysis of liquid chromatography and mass spectrometry software (2DICAL) for the identification of deregulated proteins obtained from FFPE oral squamous cell carcinoma compared to a noncancerous oral tissue.15 In the present study, we comprehensively evaluated conditions for the optimal protein extraction and separation from FFPE tissue using gel-free and gel-based proteomics. Moreover,

for the first time, the protein identifications obtained from multidimensional approaches were compared to demonstrate the most reliable and reproducible method for FFPE proteome analysis. As a model tissue, we used the heart of the C57BL/6 mouse that is easily available as both fresh/frozen and archival tissue.

2. Experimental Section 2.1. Materials. β-Octylglucoside, Triton X-100, SDS, ammonium bicarbonate, and RIPA extraction buffer were obtained from Sigma (St. Luis, MO); RapiGest from Waters; acetone, acetonitrile, formic acid, and trifluoroacetic acid (TFA) from Roth (Karlsuhe, Germany); trifluoroethanol (TFE) from Fluka (Buches, Germany); dithiothreitol (DTT), tributylphosphine (TBP), iodoacetamide, urea, thiourea, tris-(hydroxymethyl) aminomethane (Tris), CHAPS, and IPG buffer 3-10 were from GE Healthcare (Freiburg, Germany). QProteome extraction buffer was purchased from Qiagen (Germany); sequencing grade trypsin was obtained from Promega (Madison, WI); cyano-4-hydroxycinnamic acid was from Bruker Daltonik (Bremen, Germany). All solutions were prepared using HPLC grade water from Roth (Karlsuhe, Germany). 2.2. Tissue Samples. For all experiments, we used heart tissue obtained from male C57BL/6 mice. Tissues were fixed in 4% buffered formalin for 24 h, dehydrated with a graded series of ethanol before being embedded in paraffin. Blocks were stored in the dark at room temperature for between 1 and 3 years. Sections (20 µm) of the archival tissue were cut from tissue blocks after initial trimming to remove exposed surfaces. Fresh/frozen hearts used for comparison were removed and rinsed immediately with ice-cold buffer containing 20 mM KCl, 2 mM K2HPO4, 1 mM EGTA, pH 6.8, to remove excess blood. All experiments were done with three technical replicates. 2.3. Protein Extraction. FFPE mouse heart tissue sections (20 µm thick, 80 mm2 wide) were placed on microscope slides and deparaffinized by incubating twice with xylene for 10 min at room temperature before rehydration in a graded series of ethanol (100%, 95% and 70%) for 10 min each. The tissue sections were scraped from the slides, washed with 0.5% β-octylglucoside and resuspended in a range of different detergent-containing buffers including (i) Laemmli buffer containing 2% SDS,13 (ii) rehydration buffer containing 2% CHAPS,5 (iii) extraction buffer containing 0.2% Tween 20,2 (iv) RIPA buffer containing 2% SDS and 1% NP40,4 and (v) extraction buffer developed in this study containing 20 mM Tris-HCl, pH 8.8, 2% SDS, 1% β-octylglucoside, 200 mM DTT, and 200 mM glycine. All buffers contained the mixture of protease inhibitor cocktail (Complete, Roche Diagnostics). All samples were incubated in the buffers at 100 °C for 20 min, and at 80 °C for 2 h with shaking. The extracts were centrifuged for 30 min at 14 000g at 4 °C. The protein extract was precipitated with the 2D clean up kit (GE Healthcare) following the manufacturer’s instructions and protein quantification was done from the pellet resuspended in Tris buffer in triplicate using both Bradford method and 2D Quant Kit (GE Healthcare) following the manufacturer’s instructions. 2.4. Protein Separation. 2.4.1. Gel-Based Approaches. 2.4.1.1. 1D SDS-PAGE. Samples (60 µg) of the protein extract from FFPE tissue with the different buffers were solubilized in SDS-PAGE sample buffer, and separated by 12% SDS-PAGE under reducing conditions24 before staining with Brilliant blue G-250 or silver.25 Journal of Proteome Research • Vol. 9, No. 9, 2010 4711

research articles 2.4.1.2. IEF 2D SDS-PAGE. The protein extracts from FFPE and fresh/frozen material were precipitated with the 2D clean up kit (GE Healthcare) following the manufacturer’s instructions. A total of 200 µg of protein was dissolved in rehydration buffer containing 9 M urea, 4% CHAPS, 1% immobilized pH gradient buffer pH 3-10NL (GE Healthcare), and 50 mM DTT and incubated at room temperature for 1 h with shaking. The protein extracts were incubated on 13 cm 3-10NL strips O/N under low viscosity paraffin oil. Samples were separated by 2-DE IEF that was performed using an IPGphor III IEF system (GE Healthcare) at 20 °C using voltages and running times as follows: 2 h at 150 V (step), 3 h at 300 V (step), 4 h to 1,000 V (step), 2 h to 3500 (step), 2 h 10 000 V (step), and gradient to 10 000 V, hold at 10 000 V until a total of at least 45 000 V/h was reached. After focusing, the strips were equilibrated in 50 mM Tris-HCl, pH 6.8, 2% SDS, 7 M urea, 10% glycerol, reduced with 2% DTT for 15 min, and then alkylated with 2.5% iodoacetamide for 15 min. The equilibrated strips were transferred to 12% slab gels, separated in the second dimension under reducing conditions at 10 mA for 1 h and then 20 mA until bromophenol blue dye had reached the end of the gel. The gel was stained with silver.25 2.4.1.3. LIEF 2D SDS PAGE. 200 µg of FFPE protein extract was resuspended in 2.5 mL rehydration buffer containing 9 M urea, 4% CHAPS, 1% immobilized pH gradient buffer pH 3-10NL (GE Healthcare), and 50 mM DTT and incubated at room temperature for 1 h with gentle shaking and loaded into a Microrotofor cell (Bio-Rad). LIEF was performed at 1 W constant power for 3 h under denaturing conditions. Ten fractions of 200 µL each were collected, and pH and protein concentration was determined from each fraction. The fractions were precipitated O/N using cold acetone and then separated by 12% SDS gel before staining with silver.25 2.4.1.4. In-Gel Digestion for MALDI Analysis. After staining the gels, the visible protein bands or spots were excised from polyacrylamide gels and subjected to in-gel trypsin digestion before MS analysis as described26 with a slight modification as follows. The gel spots were transferred to protein low bind tubes, and destained with 15 mM K3Fe(CN)6 and 50 mM Na2S2O3. The gel pieces were washed once with 500 µL of water and afterward twice for 15 min with 200 mM NH4HCO3. The liquid was removed and the gel pieces were shrunk with 25 µL of acetonitrile (ACN) for 5 min. Subsequently, the gel pieces were dried and the samples were rehydrated with 10 µL of trypsin (Promega, 10 ng/µL in 50 mM NH4HCO3) and digested O/N at 37 °C. The resulting peptide mixture was extracted twice with 50 µL of 50% ACN, 2.5% TFA by sonication for 10 min. A third extraction was done by adding 10 µL of ACN and sonicating for 5 min. The supernatants were collected in a fresh protein low bind tube, frozen in liquid nitrogen, and reduced to a volume of 10-20 µL in a SpeedVac. The peptides were bound to C18ZipTips (Millipore) according to the manufacturer’s instructions and eluted with 50% ACN, 0.1% TFA. A total of 0.5 µL of sample was spotted onto a stainless steel MALDI target plate by the dried droplet method. The matrix used was 2.5 mg/mL R-cyano-4-hydroxycinnamic acid in 50% ACN, 0.1% TFA. 2.4.1.5. MALDI Analysis. Mass spectra were acquired using a 4700 Proteomics Analyzer (MALDI-TOF/TOF) (Applied Biosystems). Measurements were performed with a 355 nm Nb: YAG laser in positive reflector mode with a 20 kV acceleration voltage. The mass range (m/z 900-4000) was externally calibrated using the peptide calibration standard III (Applied 4712

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Azimzadeh et al. Biosystems). For each MS and MS/MS spectrum, 3000 laser shots were accumulated. Tandem mass spectrometry was performed by CID with air as collision gas. Precursor masses were selected in a data-dependent manner using the 8 most abundant ions excluding trypsin autolytic and common keratin peptide masses. Two missed tryptic cleavages were allowed and a mass accuracy of 65 ppm was used for the searches and within 0.3 u for MS/MS. Spectra acquisition and processing was done in an automatic fashion with the 4000 Series Explorer software (version 3.6, Applied Biosystems). The GPS Explorer Software (version 3.6, Applied Biosystems) was used for spectra analyses. Database search was performed with MASCOT (Version: 2.2.06) using the mouse UniRef100 version from 20090718 (selected for Mus musculus, 78 401 entries) and Swiss-Prot databases (Swiss-Prot version from 20090212). We set carbamidomethylation of cysteine as the fixed modification and oxidized methionine and deamidation of asparagine and glutamine as variable modifications. The variable 30 Da modification methylol was set for the lysine, histidine, and arginine when searching the database. Precursor tolerance was set to 75 ppm and MS/MS fragment tolerance to 0.3 Da. 2.4.2. Gel-Free Approach. 2.4.2.1. In-Solution Digestion. The extracted proteins were precipitated with cold acetone O/N, resuspended in 50 mM ammonium bicarbonate (100 mM, pH 8.0) and 0.2% RapiGest (Waters). The protein mixture was reduced with TBP (10 mM) and DTT (25 mM) at 60 °C for 30 min and alkylated with iodoacetamide (50 mM) in the dark for 30 min at room temperature before digestion with trypsin at 1:20 (w/w) O/N at 37 °C. 2.4.2.2. In-Tissue Digestion. Tryptic digestion was performed using detergent-free extraction buffer21 with a slight modification as follows. FFPE tissue was deparaffinized and rehydrated as described before and the sections were incubated in 100 µL of ammonium bicarbonate (100 mM, pH 8.0) at 80 °C for 2 h, A total of 100 µL of trifluoroethanol (TFE) was added and the sample was sonicated twice for 20 s on ice, heated for 1 h at 60 °C, and sonicated again twice for 20 s on ice. The protein mixture was reduced with TBP (10 mM) and DTT (25 mM) at 60 °C for 30 min, followed by alkylation with iodoacetamide (50 mM) for 20 min at room temperature in the dark. The reduced and alkylated sample was digested with trypsin as in section 2.4.2.1. The digested peptides from both in-solution and in-tissue digestion were lyophilized before LC MS/MS analysis. 2.4.2.3. LC-MS/MS Analysis. For the identification of proteins, SDS-PAGE bands were cut out and in-gel digestion was performed as described before. The digested peptides were separated by reversed phase chromatography (PepMap, 15 cm ×75 µm i.d., 3 µm/100 Å pore size, LC Packings) operated on a nano-HPLC (Ultimate 3000, Dionex) with a nonlinear 170 min gradient using 2% acetonitrile in 0.1% formic acid in water (A) and 0.1% formic acid in 98% acetonitrile (B) as eluted with a flow rate of 250 nL/min. The gradient settings were subsequently: 0-140 min, 2-30% B; 140-150 min, 31-99% B; 151-160 min, Stay at 99% B and equilibrate for 10 min at starting conditions. The nano-LC was connected to a linear quadrupole ion trap-Orbitrap (LTQ Orbitrap XL) mass spectrometer (ThermoFisher, Bremen, Germany) equipped with a nano-ESI source. The mass spectrometer was operated in the data-dependent mode to automatically switch between Orbitrap-MS and LTQ-MS/MS acquisition. Survey full scan MS

FFPE Proteome Analysis Using Gel-Free and Gel-Based Proteomics spectra (from m/z 300 to 1500) were acquired in the Orbitrap with resolution R ) 60 000 at m/z 400 (after accumulation to a target of 1 000 000 charges in the LTQ). The method used allowed sequential isolation of the most intense ions, up to 10, depending on signal intensity, for fragmentation on the linear ion trap using collision-induced dissociation at a target value of 100 000 ions. High resolution MS scans in the Orbitrap and MS/MS scans in the linear ion trap were performed in parallel. Target peptides already selected for MS/MS were dynamically excluded for 30 s. General mass spectrometry conditions were: electrospray voltage, 1.25-1.4 kV; no sheath and auxiliary gas flow. Ion selection threshold was 500 counts for MS/MS, and an activation Q-value of 0.25 and activation time of 30 ms were also applied for MS/MS. All MS/MS data were analyzed using MASCOT (Matrix Science) Version: 2.2.06 and Sequest (ThermoFinnigan, San Jose, CA; version SRF v. 5). Sequest was set up to search the mouse.fasta database UniRef100 20090718 (8 663 575 sequences; 3 067 231 997 residues) and Swiss-Prot 20090708 (495 880 sequences; 174 780 353 residues) assuming the digestion enzyme trypsin and with a fragment ion mass tolerance of 1 Da and a parent ion tolerance of 10 PPM. Iodoacetamide derivative of cysteine as stable and oxidation of methionine, deamidation of arginine and glutamine as well as hydroxyl modification of lysine, arginine, and histidines as variable modifications were specified in Sequest and MASCOT as variable modifications. Scaffold (version Scaffold_2_02_03, Proteome Software, Inc., Portland, OR) was used to validate MS/MS-based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 80.0% probability as specified by the Peptide Prophet algorithm.27 Protein identifications were accepted if they could be established at greater than 95.0% probability and contained at least 2 identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm.28 Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. 2.5. Immunoblot Analysis. Proteins separated by SDS-PAGE were transferred to nitrocellulose membranes (GE Healthcare) using a TE 77 semidry blotting system (GE Healthcare) at 1 mA/cm for 1 h .29 The membranes were blocked using 3% BSA in PBS, pH 7.4, for 1 h at room temperature, washed three times in 10 mM Tris-HCl, pH 7.4, 150 mM NaCl for 5 min, and incubated O/N at 4 °C with antibodies against actin (Santa Cruz Biotechnology), cardiac heavy chain myosin (Affinity Bioreagent), 14-3-3 protein (Abcam), or tubulin (Sigma Aldrich) using dilutions recommended by the manufacturer. After washing three times as described above, the blots were incubated with horseradish peroxidise-conjugated anti-mouse, anti-rabbit, or anti-goat secondary antibody (Santa Cruz Biotechnology) for 1 h at room temperature and developed using the ECL system (GE Healthcare) following standard procedures. Data Availability. Access to raw MS data is provided in PRIDE (http://www.ebi.ac.uk/pride). Here the PRIDE XML files converted with the PRIDE converter tool from the Mascot.dat files for each MALDI spot of all relevant experiments for this publication can be found. Accession numbers: 12227-12237. Reviewer account: Username, review72549; Password, gt9wNc_2.

3. Results and Discussion 3.1. Extraction of Proteins from FFPE Heart Tissue. To establish an optimized extraction method for FFPE samples,

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we used C57BL/6 murine heart tissue as a model system. For this purpose, we used both archival samples and fresh/frozen tissue for comparison. To test the reproducibility and effectiveness of protein extraction, different combinations of detergents, denaturants, reductants, pH values, and temperatures were examined on the same batch of tissue material. The protein yield and the 1D pattern using different extraction buffers are shown in Figure 1. The extraction buffer developed for this study (Figure 1, lane l) resulted in maximal protein yield and the clearest band pattern when compared to other buffers published previously (Figure 1, lanes a-k). To scavenge cross-linking, we used glycine in our extraction buffer in a concentration of 200 mM. Treatment of archival tissue with buffers containing glycine often resulted in extra bands and a smear in low molecular weight region (Figure 1, lanes e-i). However, this was not the case when glycine was used as a component of our buffer (lane l). It has been reported that protein recovery after extraction is affected by pH and it therefore needs to be adjusted based on the tissue type.11 In this study, maximal yield was obtained by using a basic Tris buffer (pH 8.8). Incubation of the FFPE section with acidic buffer (pH 4) generated a smeared protein pattern after separation (Figure 1, lanes e and h). Most of the proteomics studies on FFPE have used extraction buffers containing SDS4,11,13,30 due to its functions as a detergent and a protein denaturant. We showed that a mixture of nonionic detergent (1% β-octylglucoside) together with 2% SDS resulted in optimal protein release from FFPE sections; increasing the amount of SDS (>4%) did not enhance the protein yield further, on the contrary, it generated a diffused protein pattern on 1DE (not shown). Treatment of FFPE tissue with disulfide reducing agents such as DTT, TBP, and β-mercaptoethanol during extraction resulted in an enhanced protein recovery. However, we discovered that the majority of the detected proteins remained as cross-linked protein complexes (see below), indicating that extraction in reducing conditions did not efficiently reverse the intra- and intermolecular cross-linking resulting from formalin fixation. The optimal extraction conditions were incubation under constant shaking at 100 °C for 20 min followed by incubation at 80 °C for 2 h. The benefit of using high temperatures for the proteinextractionfromFFPEtissuehasbeenshownpreviously.6,7,12 Heating may renature the cross-linked proteins by a partial thermal hydrolysis of methylene bridges.23,31 The average protein yield from a 20 µm thick, 80 mm2 wide tissue slice was 48 µg/slice, a yield significantly greater than when using the commercial buffer (25 µg/slice). The extraction efficiency is demonstrated in Figure 2 where, after the first protein extraction using our buffer (lane b), the insoluble material was resuspended in the same buffer and separated with 1D-PAGE (lane c). Lane c had no significant protein content, indicating an efficient extraction was completed in the first step. An additional washing step using a low concentration (1%) of β-octylglucoside improved the resolution of the 1D pattern. The analysis of the supernatant of this washing step by 1D gel showed no protein bands, suggesting that proteins were not released during the washing (Figure 2, lane a). 3.2. 1DE Protein Separation and Protein Identification. To analyze the quality of the electrophoretic separation using 1D SDS-PAGE, the patterns of FFPE and fresh/frozen tissue were compared (Figure 2, lanes b and d). Although we have shown Journal of Proteome Research • Vol. 9, No. 9, 2010 4713

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Figure 1. Comparing the electrophoretic pattern of extracted protein using different detergent containing buffers. Proteins from FFPE mouse heart were extracted using different extraction buffers and 20 µg of FFPE extracted proteins was separated on 12% 1D-PAGE under reducing condition before staining by coomassie (lines a-l); Tris buffer containing 1% β-octylglucoside (a), rehydration buffer containing 2% CHAPS (b), Laemmli buffer containing 2% SDS (c), RIPA buffer containing 2% SDS and 1% NP40 (d), acidic Tris buffer containing Glycine, 2% SDS (e) or 0.2% Tween 20 (h), neutral Tris buffer containing Glycine, 2% SDS (f) or 0.2% Tween 20 (g), basic Tris buffer containing 2% SDS (or 0.2% Tween 20 and Glycine with DTT (i) and without DTT (j), commercial extraction buffer (k) and extraction buffer used in this study containing 20 mM Tris-HCl, pH 8.8, 2% SDS, 1% β-octylglucoside, 200 mM DTT, 200 mM glycine and a mixture of protease inhibitor cocktail (l). The numbers indicate the gel slices were cut for ESI-LC-MS/MS analysis.

(Figures 1 and 2) that using the buffer developed in this study results in a relatively clear 1DE pattern and efficient extraction of archival tissue proteins, the quality and the quantity of the proteins are clearly inferior when compared to those of fresh/ frozen tissue. Clearly visible bands migrating in the molecular weight range 10-150 kDa were present in both fresh/frozen and FFPE sampes on silver-stained 1D SDS-PAGE, but most of the protein bands from FFPE tissue appeared less sharp than those from fresh/frozen tissue (Figure 2, lanes b and d). An intense signal in the higher molecular weight region suggested the accumulation of cross-linked proteins. The low quality of the FFPE protein pattern separated on SDS-PAGE has been reported previously.13,23,30 However, in contrast to previous studies where no intact molecular mass bands were observed, we were able to show distinct bands even in the high molecular weight region. To determine the integrity and compatibility of the FFPE extracted proteins with mass spectrometry approach, the 1 DE gel, consisting of protein bands in the range of 10-200 kDa, was cut into 17 slices (Figure 1) and digested with trypsin. The fractions were analyzed by LC-MS/MS as described in section 2.4.2.3. A total of 1312 unique peptides were isolated; based on them, 192 proteins were identified by two or more peptides (Supplementary Table 1). The number of identified proteins could be increased significantly by manually validating the data of the proteins identified by a single peptide. An example of this is the 14-3-3-like protein. Although this protein was identified by only one peptide, the MS/MS analysis showed good sequence coverage of the y and b ion series (see Supporting Information). The theoretical molecular weight of the identified proteins ranged from 8 kDa (mitochondrial ATP synthase subunit e) up to 312 kDa (fibrillin 1). The isoelectric point (pI) values ranged 4714


from 4.09 (calsequestrin) to 12.02 (ribosomal protein L28). Approximately 75% of the identified proteins had a pI between 4 and 7. Only 18% of the identified proteins migrated in a single fraction corresponding to the predicted molecular weight (Figure 3, lane 1), suggesting that they were in an intact monomeric form. The remaining (82%) were present either as higher molecular weight moieties, suggesting formaldehyde cross-linking, or were smaller than predicted, indicating protein fragmentation due to heating, aging, or possible contact with proteases during the fixation and embedding (Figure 3, lanes 2-17). The MS/MS analysis showed that the high molecular weight region of the 1D gel was enriched for identified peptides originating from proteins with unexpected molecular weight. However, the “top hit” identified proteins in each fraction migrated with the correct molecular weight. Peptides originating from the most abundant proteins such as actin, ATP synthase subunits, and albumin, or contaminating proteins such as hemoglobin and keratin, were found in all 17 fractions (Figure 3, lane 17). Tandem mass spectrometry showed that in most of the cases the number of identifications corresponding to the number of identified spectra was considerably lower than 30%, indicating the low intensity of the detected peaks. This is a general problem originating from the low quality of the FFPE sample and may not be totally overcome by any measures. 1DE fractions 4-9 corresponding to the 50-90 kDa molecular regions resulted in quantitatively and qualitatively best proteins identifications by MS/MS analysis. Krause et al. observed a general preferential detection of tryptic peptides with the C-terminal arginine over lysine in MALDI MS.32 In FFPE tissue, the percentage of lysine residues is even lower (about 10%) than that found in fresh/frozen tissue.18 In good agreement with previously published data using FFPE,18,21 a comparison of the identified peptides

FFPE Proteome Analysis Using Gel-Free and Gel-Based Proteomics

Figure 2. The protein extraction efficiency of evaluated extraction buffer. The 20 µm thick, 80 mm2 wide FFPE tissue slice was deparaffinized and washed gently using low concentration (1%) of the detergent β-octylglucoside before incubation with extraction buffer. The supernatant of the washing step (a), extracted proteins (b), and final insoluble pellet (c) were separated by 1DE and compared to fresh/frozen tissue extract (d). The same amount of protein was loaded on the gel in all cases.

Figure 3. Distribution of the identified proteins in multiple slices. The numbers above the columns represent the identified proteins found in the given number of slices, where column (1) represents proteins identified in single slice and (17) shows the proteins present in all 17 slices.

indicated that the C-terminal arginine-containing peptides were detected more frequently than the lysine-ending peptides. The low number of detected lysine-ending peptides in our study may indicate that modified lysine residues are not accessible to trypsin, probably due to the involvement of the lysine containing peptides in the cross-linking.

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To answer this question, the variable 30 Da modification methylol caused by the cross-linking11,33,34 was set for the lysine, histidine, and arginine during searching against the database. The reanalysis of the generated peptides from 1DE revealed that only 4.5% of all identified peptides contained modified lysine residues. This result is consistent with the previously published data showing that the majority of detected peptides contained no modified lysine residues.18,21 Our study shows for the first time the composition of the modified amino acid containing peptides from FFPE proteins (Table 1). Manual spectral evaluation of these peptides showed that they belong to 6 proteins: actin (5 peptides), albumin and myoglobin (2 peptides each), hemoglobin subunit alpha and beta, keratin and myosin regulatory light chain (one peptide each). The distribution of the detected modified peptides through the 1D gel fractions is demonstrated in Table 2. Since the majority of the modified peptides belonged to actin, amino acid compositions of 4 modified peptides from this protein were subjected to further analysis. As shown in Table 3, all detected modified lysine-containing peptides from actin have at least one missed cleavage site indicating that the modification of a lysine residue prevents cleavage by trypsin at that site. This finding is consistent with the low number of identified lysine-ending peptides. The unmodified lysine residues were detected in shorter peptides without missed cleavage site. Analysis of the peptides revealed that, when lysine was modified, the neighboring methionine was not oxidized, probably due to steric hindrance. Only one peptide containing modified histidine was identified. Modified arginine residues were not detected, which is consistent with the high frequency of unmodified arginine-ending peptides found. 3.3. Immunoblotting on the FFPE Extracted Proteins. Most of the previous studies report the suitability of FFPE tissue extraction buffers for immunoblotting systems.7,30 To validate the extraction buffer developed in this study, we performed immunoblotting using antibodies against abundant identified proteins such as tubulin, actin, and cardiac heavy chain myosin (Figure 4, lanes a-h). Although there was a large difference in the electrophoretic pattern between FFPE and fresh/frozen extracts (Figure 2, lanes b and d), no significant differences in the migration behavior and reactivity of the intact proteins could be seen after Western blotting. The comparison of Western blotting of FFPE and fresh/frozen samples showed that the main bands, corresponding to intact proteins, migrated with the expected molecular weights (Figure 4, lanes a-h), suggesting that cross-linking was to some extent reversible in the extraction conditions used. However, as shown in Figure 4 (lanes b and d), actin and myosin showed multiple additional bands in FFPE extracts, indicating an irreversible cross-linking reaction. This is consistent with our observation that many peptides were found in fractions not corresponding to the right molecular weight of the protein (Figure 3 and Table 2). In contrast to a previous study that failed to detect heavy chain myosin using FFPE extract,30 we found the main signal at the expected molecular weight (Figure 4, lane b). In the case of tubulin, a single band of 50 kDa was detected in both FFPE and fresh/frozen samples (Figure 4, lane e and f). The results were consistent with MS data showing that most of the identified peptides from tubulin were found in the fraction corresponding to the expected molecular weight. To test the ability to find low-abundance proteins and the ability to identify proteins based on only one peptide, we Journal of Proteome Research • Vol. 9, No. 9, 2010 4715

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Azimzadeh et al. a

Table 1. The Sequences of the Modified Peptides after 1DE ESI LC-MS/MS

sequence

MASCOT MOWSE ion score

MASCOT identity score

observed mass

actual mass

charge

(K)YPIEHGIITNWDDMEK(I) (K)EITALAPSTMKIK(I) (R)MQKEITALAPSTMK(I) (R)HQGVMVGMGQKDSYVGDEAQSK(R) (R)DLTDYLMKILTER(G) (K)ATAEQLKTVMDDFAQFLDTCCK(A) (R)ENYGELADCCTK(Q) (R)YFDSFGDLSSASAIMGNAK(V) (R)MFASFPTTKTYFPHFDVSHGSAQVK(G) (R)HSGDFGADAQGAMSK(A) (K)HGCTVLTALGTILK(K) (K)EAPGPINFTVFLTMFGEK(L) (R)TAAENEFVGLK(K)

28.0 31.0 59.0 32.0 46.0 29.0 42.0 98.0 35.0 86.0 34.0 40.0 44.0

33.0 29.0 33.0 32.0 33.0 24.0 33.0 33.0 35.0 28.0 30.0 33.0 33.0

664.3128 716.9082 797.9133 799.6987 820.9328 745.3006 874.7348 1,005.96 954.131 754.826 757.4252 1,014.51 604.81

1989.9151 1431.8008 1593.811 2396.0728 1639.85 1488.5856 2621.1811 2009.9054 2859.3697 1507.6364 1512.8348 2027.0094 1207.6078

3 2 2 3 2 2 3 2 3 2 2 2 2

protein name

Actin

Albumin Hemoglobin subunit beta-1 Hemoglobin subunit alpha Myoglobin Myosin regulatory light chain 2 Keratin, type cytoskeletal 2 a

The modified amino acids are shown in bold.

Table 2. Distribution of the Modified Amino Acids Containing Peptides in 17 1DE Slicesa slice no.

proteins description

accession number

Mw (kDa)

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

1 2 3 4 5 6 7

Actin Hemoglobin subunit alpha Hemoglobin subunit beta-1 Keratin, type II cytoskeletal 2 Myoglobin Myosin regulatory light chain 2 Serum albumin

Q3TG92 Q91VB8 A8DV59 P04104 P04247 P51667 P07724

42 15 16 63 17 19 69

0 0 1 0 0 0 0

0 0 0 0 0 0 0

1 0 1 0 1 0 0

4 0 1 1 0 0 0

5 0 1 1 0 0 2

0 0 0 0 0 0 0

1 0 1 0 0 0 0

6 0 0 0 0 0 0

2 0 0 0 0 0 0

0 0 0 0 0 0 0

0 0 0 0 0 0 0

0 0 0 1 0 0 0

1 1 1 0 0 0 0

0 0 1 0 0 0 0

0 0 0 0 0 1 0

1 0 0 1 0 0 0

0 0 0 1 1 0 0

a

The number of unique peptides containing the modified amino acids is shown.

Table 3. The Identified FFPE Heart Proteins after IEF-2DE-MALDI-MS/MS Analysisa no.

protein description

accession number

Mw (kDa)

protein score

pI

sequence coverage

uinque peptides matched

1 2 3 4 5 6 7 8 9

Actin alpha Alpha-Crystallin B chain ATP synthase subunit alpha ATP synthase subunit beta ATP synthase subunit O Hemoglobin subunit beta-1 Myosin light chain 3 Myosin regulatory light chain 2 Troponin Isoform A1

Q3TG92 P23927 Q03265 P56480 Q9DB20 A8DV59 P09542 P51667 P50752

42 20 55 56 23 15 225 18 34

198 110 131 244 790 59 283 102 118

5.23 6.76 9.22 5.19 10.00 7.9 5.03 4.86 5.16

19% 29% 16% 24% 29% 21% 43% 27% 22%

7 8 7 13 10 5 11 13 7

a

Theoretical isoelectric points and molecular weights are derived from the amino acid sequences in Swiss-Prot.

validated the MS/MS data of 14-3-3-like protein by Western blotting. The immunoblotting detected a single signal of 27 kDa corresponding to the expected molecular weight in both FFPE (Figure 4, lane g) and fresh/frozen (lane h) protein extracts. This confirmed the compatibility of the extraction method and the subsequent mass spectrometry strategy for identification of very low abundance proteins. 3.4. Separation and Identification of Proteins by IEF2DE. Although previously not proven to be a successful method to analyze FFPE material, conventional 2D electrophoresis for protein separation was used as a comparison with other methods. Figure 5a shows that only a few spots ranging from 20 to 80 kDa were detected on the 2D-PAGE gel after silver staining. Most of the detected spots were migrating in the acidic area, while the basic proteins were missing in comparison to the fresh/frozen tissue (Figure 5b). Thirty-four spots were excised and subjected to MALDI MS/MS identification after ingel digestion. Mass spectrometry analysis identified 9 proteins (Table 4). Intense protein spots were identified at the correct 4716


molecular weight and pI. Five identified proteins were present in multiple spots (a total of 15 spots) showing the potential protein modifications. The comparison with previously published results using 2DEPAGE to analyze FFPE material shows that the extraction buffer used in this study improves the separation; it has been shown that proteins extracted by rehydration buffer containing urea and CHAPS were not resolved and no clear spot pattern was detected.5,9,10 Hood et al. concluded that the classical 2DE methodology for proteins extracted from FFPE tissue yielded essentially no results.22 Our study showed a comparable pattern to that published recently by Addis et al.14 However, as we show clear benefits from other strategies, we suggest that classical 2DPAGE is not the optimal method for a proteomic analysis of FFPE. 3.5. Separation and Identification of Proteins by LIEF2DE. The lack of basic proteins on 2D gel samples may be caused by insolubility of the protein extract, a high percentage of detergent in the sample, and/or incomplete isoelectric focusing in the strip. To test the latter, the extracted proteins

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FFPE Proteome Analysis Using Gel-Free and Gel-Based Proteomics

Figure 4. Immunoblotting analysis of FFPE extracted proteins. The extracted proteins from fresh/frozen (lanes a, c, e, and g) and FFPE (lines b, d, f, and h) tissue samples were separated by 12% SDS-PAGE and analyzed by immunoblotting using anti-myosin (a and b), anti-actin (c and d), anti-tubulin (e and f), and anti-14-3-3(g and h). The same amount of protein was used in all cases.

Figure 5. Comparing of the 2D electrophoretic pattern of FFPE extracts using different focusing methods. A total of 200 µg of total extracted proteins from FFPE samples was focused on pH gradient 3-10 NL using either IEF (a) or LIEF (c) and separated on 2D PAGE. Protein detection was performed by staining with silver. The FFPE electrophoretic patterns were then compared with 200 µg of the fresh/frozen tissue extracts (b) focused on 3-10 NlL strip and separated under the identical condition. Table 4. The Identified FFPE Heart Proteins after LIEF-1DE-MALDI-MS/MS Analysisa no.

protein description

accession number

Mw (kDa)

protein score

pI

sequence coverage

unique peptides matched

1 2 3 4 5 6 7

Actin alpha ATP synthase subunit alpha ATP synthase subunit beta Hemoglobin subunit beta-1 Keratin, type II cytoskeletal Myosin light chain 3 Serum albumin

Q3TG92 Q03265 P56480 A8DV59 P04104 P09542 P07724

42 59 56 15 60 225 70

423 138 523 59 520 140 900

5.23 9.22 5.19 7.9 8.51 5.03 5.75

63% 44% 45% 21% 32% 67% 9%

32 26 30 5 14 28 6

a


were focused in liquid phase. The proteins were separated in 10 fractions by LIEF and stained with silver (Figure 5c). The overall pattern of focused proteins was comparable to that of the 2D PAGE, the proteins mainly being detected in the acidic

fractions. The accumulation of the proteins on the top of the each lane suggested precipitation of protein complexes. The visible bands from LIEF-1D PAGE were analyzed by MALDI MS/MS. Twenty-four bands were cut for the tryptic cleavage Journal of Proteome Research • Vol. 9, No. 9, 2010 4717

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Azimzadeh et al. a

Table 5. The Shared Identified FFPE Heart Proteins after In-Solution and In-Tissue Digestion

identified unique peptides in-solution digestion

protein description

accession number

Mw (kDa)

1 2 3 4 5 6 7 8 9 10 11 12 13

2-oxoglutarate dehydrogenase E1 component, mitochondrial Acetyl-Coenzyme A acyltransferase 2 Aconitate hydratase, mitochondrial Actin alpha Actinin alpha 2 ADP/ATP translocase 1 ATP synthase subunit alpha, mitochondrial ATP synthase subunit beta, mitochondrial Citrate synthase, mitochondrial Cytochrome b-c1 complex subunit 2, mitochondrial Cytochrome c oxidase subunit 6B1 Desmin n ) 2 Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex, mitochondrial Electron transfer flavoprotein subunit alpha, mitochondrial Enoyl-CoA hydratase/isomerase Fatty acid-binding protein, heart Hemoglobin subunit alpha Hemoglobin subunit beta-1 Histone H2A type 1-F Histone H2B type 1-F Isocitrate dehydrogenase [NADP], mitochondrial Long-chain specific acyl-CoA dehydrogenase, mitochondrial Malate dehydrogenase, mitochondrial M-protein Myoglobin n ) 2 Myosin binding protein C, cardiac Myosin light chain 3 Myosin regulatory light chain 2, ventricular/cardiac muscle isoform Myosin-6 Myozenin-2 NADH dehydrogenase (Ubiquinone) 1 alpha subcomplex NADH dehydrogenase [ubiquinone] iron-sulfur protein 3, mitochondrial NADH-ubiquinone oxidoreductase 75 kDa subunit Putative uncharacterized protein Sarcoplasmic/endoplasmic reticulum calcium ATPase 2 Serum albumin Succinate dehydrogenase [ubiquinone] flavoprotein subunit, mitochondrial Triosephosphate isomerase Tropomyosin1 alpha Troponin T, cardiac muscle Tubulin

Q60597 Q3TIT9 Q99KI0 Q3TG92 Q8K3Q4 P48962 Q03265 P56480 Q9CZU6 Q9DB77 P56391 P31001 Q8BMF4

116 42 85 42 104 33 60 56 52 48 10 53 68

6.36 8.33 8.08 5.23 5.34 9.73 9.22 5.19 8.72 9.26 8.96 5.21 8.81

3 4 4 7 8 3 6 7 3 1 2 3 2

1 3 2 6 3 4 11 9 1 2 0 3 0

Q99LC5 Q8BH95 P11404 Q91VB8 A8DV59 Q64426 P10853 P54071 P51174 P08249 O55124 P04247 A2AGQ1 P09542 P51667

35 50 15 15 16 14 14 51 48 36 165 17 141 22 19

8.62 9.61 6.11 7.97 7.85 11.05 10.31 8.88 8.53 8.93 5.51 7.06 6.18 5.03 4.86

2 2 2 1 4 1 0 4 3 3 2 3 3 4 2

3 0 1 2 5 2 2 5 3 7 0 5 0 4 1

Q02566 Q9JJW5 Q6GTD3 Q9DCT2

224 30 43 30

5.57 8.53 9.75 6.67

32 2 2 2

9 0 1 0

Q3TIU7 Q9D6U7 O55143 P07724 Q8K2B3

80 43 115 69 73

5.51 6.58 5.23 5.75 7.06

2 5 2 4 2

0 0 0 3 2

P17751 Q8BP43 P50752 Q3TIZ0

27 33 36 50

6.9 4.69 4.98 4.96

2 2 0 2

0 2 3 0

14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 a


and 7 proteins were identified with significant scores (Table 5). Mass spectrometry analysis showed that a significant number of peptides generated from abundant proteins such as actin subunits and ATP synthase subunit alpha were identified several times in unexpected positions. Proteins such as albumin, myosin light chain, and keratin were found in expected molecular weight and pI but with low sequence coverage. Most of the protein identifications could be verified only by single MS/MS, indicating the poor quality and quantity of the FFPE samples. The absence of the basic proteins on both 2D PAGE and LIEF strongly suggests the involvement of basic amino acid containing proteins in the cross-linking reaction. We reanalyzed the MS data of peptides originating from actin and appearing at unexpected molecular weight regions using LIEF-1DE. The methylol modification was used as described 4718

pI

in-tissue digstion

no.


above. The analysis confirmed that the same modified lysine residues were detected as in 1DE analysis (not shown). The separation of FFPE proteins using LIEF is presented for the first time in this study. However, the low resolution of the protein bands makes LIEF-2DE a suboptimal separation method for this material. Furthermore, MALDI/TOFMS analysis in both strip and liquid focusing approaches showed that the intensity of low-abundance proteins was suppressed by the presence of dominant peptides of abundant proteins making the use of MALDI-TOF/TOF for FFPE protein identification problematic. 3.6. Protein Identification with Gel-Free Approach. We used gel-free proteomics performing either in-tissue or insolution tryptic digestion. The tryptic peptides thus generated were analyzed by LC-MS/MS. The numbers of proteins identi-

FFPE Proteome Analysis Using Gel-Free and Gel-Based Proteomics fied were 111 and 93 for the in-tissue and in-solution digested samples, respectively. Almost one-third of the proteins were identified by at least two peptides; 45% of identified proteins overlapped between the two approaches (Table 5). The slightly higher number of proteins identified after in-tissue digestion may result from disturbance by detergents in the case of the in-solution digestion. Several factors such as denaturation, solubility of the proteins, and accessibility of tryptic sites may influence the number of identified peptides when using either in-tissue or in-solution digestion. The question which method is superior may also be tissue-specific. Again, less peptides with a C-terminal lysine residue were detected in comparison to arginine-ending peptides. Analysis of the MS/MS data showed that 45% of all detected spectra resulted in a protein identification. The theoretical molecular weight of the identified proteins varied between 10 kDa (cytochrome C oxidase) and 224 kDa (myosin). The pI values ranged from 4.7 (tropomyosin 1 alpha) to 11.05 (histone H2A type 1-F). The number of proteins identified by gel-free approach was smaller than by 1DE combined with LC-MS/MS, the gel-based separation step presumably reducing the complexity of the sample in a beneficial manner. The gel-free approach could equally benefit from the introduction of an additional fractionation step such as Reverse phase or Strong Cation Exchange chromatography preceding the mass spectrometry analysis. Recently, several studies have been published using in-tissue digestion with a commercial extraction buffer; most of these studies applied sophisticated downstream mass spectrometry methods to identify FFPE peptides.15,20,22,23 Although these gelfree proteomic studies provided a large number of identified proteins, most identifications were based on a single peptide assignment22,23 or used nonarchival formalin-fixed samples without paraffin embedding,18,22 all these parameters probably influencing the number of identified proteins in a positive manner. 3.7. Overlapping of Protein Profiles Using Different Separation Methods. The Venn diagram shown in Figure 6 demonstrates the overlap among of the proteins identified by the different approaches. Only proteins identified by at least two peptides are shown in the figure. The largest number of proteins was identified by LC-MS/MS after 1DE separation of proteins. Almost all of the identified proteins using other methods were also found by the 1DE/LC-MS/MS approach. Five abundant proteins, namely, actin, ATP synthase alpha and beta subunits, hemoglobin subunit beta, and myosin light chain were identified by all methods. All proteins identified by IEF2DE were also detected by other methods.

4. Conclusions This study provides extraction and separation methods for FFPE material that are compatible with downstream proteomics analyses. The protocol validated in this study using different gel-free and gel-based approaches enables highly efficient extraction of proteins and peptides from FFPE tissues, generates reproducible protein patterns, and indicates potential modification sites on the peptides. We confirm previous results showing that LC/MS based methods are in general superior to 2D/MS based methods. The study presents evidence of cross-linked complexes, consisting especially of basic proteins, and protein fragmentation that for the first time gives an explanation to problems seen with 2D gel-based methods. We have developed an optimized extraction

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Figure 6. Venn diagram of proteins identified from different FFPE heart protein separations by different approaches. The proteins used to provide the diagram were identified by two or more peptides.

procedure using a new buffer in combination with 1D and LC/ MS; this resulted in the greatest number of peptides identified. Differences in the FFPE tissue, experimental procedure, and downstream mass spectrometry approaches make direct comparisons between published studies difficult. Nevertheless, the easy-to-use, reproducible, rapid and relatively inexpensive extraction and separation method presented here is, considering the number of reliably identified proteins in the FFPE material, comparable with the best data published so far. Abbreviations: ESI-MS/MS, electrospray ionization tandem mass spectrometry; HPLC, high performance liquid chromatography; IEF, isoelectric focusing; LIEF, liquid isoelectric focusing; MS; mass spectrometry; MALDI TOF/TOF, matrix assisted laser desorption/ionisation time of flight; Mw, Molecular weight LC-MS, liquid chromatography-mass spectrometry; SDS, sodium dodecyl sulphate; O/N, overnight; pI, isoelectric point.

Acknowledgment. The research leading to these results is supported by a grant from the European Community’s Seventh Framework Programme (EURATOM) contract no. 232628 (STORE). Supporting Information Available: The identified FFPE heart proteins after 1DE-ESI-LCMS/ MS analysis; MS/ MS analysis of peptide 1532.7 from 14-3-3 protein. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Tapio, S.; Atkinson, M. J. Molecular information obtained from radiobiological tissue archives: achievements of the past and visions of the future. Radiat Environ Biophys 2008, 47 (2), 183–7. (2) Aerni, H. R.; Cornett, D. S.; Caprioli, R. M. High-throughput profiling of formalin-fixed paraffin-embedded tissue using parallel electrophoresis and matrix-assisted laser desorption ionization mass spectrometry. Anal. Chem. 2009, 81 (17), 7490–5. (3) Conti, C. J.; Larcher, F.; Chesner, J.; Aldaz, C. M. Polyacrylamide gel electrophoresis and immunoblotting of proteins extracted from paraffin-embedded tissue sections. J Histochem Cytochem 1988, 36 (5), 547–50.

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