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Development of Efficient Protein Extraction Methods for Shotgun Proteome Analysis of Formalin-Fixed Tissues

Xiaogang Jiang,, Xinning Jiang, Shun Feng, Ruijun Tian, Mingliang Ye,*, and Hanfa Zou*,
National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China, and School of Medicine, Suzhou University, Suzhou, Jiangsu 215007, China
Received October 10, 2006

There are vast archives of formalin-fixed tissues spanning many conceivable conditions such as different diseases, time courses, and different treatment and allowing acquisition of the necessary numbers of samples to carry out biomarker discovery study. However, the conventional protein analysis approach is not applicable for the analysis of proteins in the formalin-fixed tissue because the formalin fixation process resulted in the cross-linking of proteins, and thus, intact proteins cannot be efficiently extracted. In this study, several protocols were investigated to extract proteins from formalin-fixed mouse liver tissue for shotgun proteome analysis. It was found that incubation of tissue in a lysis buffer containing 6 M guanidine hydrochloride at high temperature led to the highest protein yield and the largest number of proteins identified. The peptides and proteins identified from formalin-fixed tissue were first comprehensively compared with those identified from frozen-fresh tissue. It was found that a majority of peptides identified from fixed tissue were unmodified and proteome coverage for the analysis of fixed tissue was not obviously compromised by the formalin fixation process. Valuable proteome information could be obtained by shotgun proteome analysis of formalin-fixed tissue, which presents a new approach for disease biomarker discovery.
Keywords: formalin-fixed tissue protein extraction shotgun proteome tandem mass spectrometry

Introduction
Tissue-based proteome studies promise to decipher proteome complexities of the tissue microenvironment and deliver biomarker information with appropriate pathologic and histologic relevance.1-5 Mass spectrometric profiling of complex cellular proteome obtained from diseased tissue has previously been demonstrated with frozen cancer tissue.6-9 Though fresh and /or frozen tissue samples represent attractive samples for biomarker discovery, the use of frozen tissue for such analysis has some disadvantages. Biomarker discovery typically requires analysis of dozens of different samples; however, the collection of enough numbers of these tissue samples is very difficult especially for human tissue. The frozen tissue also requires specialized equipment for storage, which is expensive and not suitable for long time storage. In contrast, formalin fixation of tissue is a standard processing methodology practiced in medical laboratories worldwide resulting in a highly stable form of tissue that is easily stored due to its inherent stability at room temperature. This routine process provides an easily stored archive of tissue that is physiologically/pathologically welldefined.
* Authors for correspondence. National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China. Prof. Dr. Hanfa Zou: tel, +86-411-84379610; fax, +86-41184379620; e-mail, hanfazou@dicp.ac.cn. Dr. Mingliang Ye, tel, +86-41184379620; fax, +86-411-84379620; e-mail, mingliang@dicp.ac.cn. Dalian Institute of Chemical Physics, Chinese Academy of Sciences. Suzhou University.

However, the formalin fixation process leads to the formation of a net of covalent cross-links between side chains of proteins by methylenic bridge formation. Therefore, methods for analysis of proteins in formalin-fixed tissue are limited to immunohistochemistry (IHC),10 a technique that provides the intracellular localization of a protein. Unfortunately IHC requires a priori knowledge of individual proteins being analyzed, and it is not a high-throughput protein analysis approach for largescale analysis. Conventional protein analysis approaches are not applicable to formalin-fixed tissue because the intact proteins cannot be efficiently extracted. Development of strategies to permit utilization of the universal formalin-fixed specimens will be important in leveraging the application of powerful mass spectrometry based proteomic approaches into the investigation of archival experiment/clinical specimens. The shotgun proteomics strategy, based on digesting proteins into peptides and sequencing them using tandem mass spectrometry and automated database searching, has become the method of choice for identifying proteins in most large-scale studies.10-13 Several groups have reported the application of the shotgun method to analyze the proteome of formalin-fixed specimens. Prieto et al.14 and Hood et al.15 reported the use of commercial Liquid Tissue buffer to extract proteins from formalin-fixed, paraffin-embedded (FFPE) tissues for shotgun proteome analysis. Hundreds of proteins from formalin-fixed prostate cancer tissue were successfully identified, including several known prostate cancer markers such as prostate10.1021/pr0605318 CCC: $37.00 2007 American Chemical Society

1038

Journal of Proteome Research 2007, 6, 1038-1047

Published on Web 02/01/2007

Shotgun Proteome Analysis of Formalin-Fixed Tissue

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HPLC grade) was from Merck (Darmstadt, Germany). Cyanogen bromide (CNBr) was purchased from Shenyang Chemical Factory (Shenyang, China). C-57BL/6J mouse was from Dalian Medical University. The mouse liver was fixed in 10% formalin for 2 weeks,21-23 and the fresh mouse liver was stored at -80 C. Protein Extraction and Proteolysis. Protein concentration was determined by the Bradford assay. Unless otherwise stated, the resulting tryptic digests were desalted with a C18 solidphase cartridge. Five different protocols were used to extract proteins from mouse liver tissues; the first protocol was applied to both frozen fresh tissue and formalin-fixed tissue, and the remaining four protocols were applied only to formalin-fixed tissue. The detailed procedures were as follows: (1) For the protocol using 6 M guanidine-HCl without heating, mouse liver tissue, either frozen or formalin-fixed tissue, was homogenized in lysis buffer (40 mM Tris, 6 M guanidine-HCl, and 65 mM DTT, pH 8.2) and then sonicated for 180 s followed by centrifugation at 25 000g for 1 h. The supernatant was collected as protein sample A for frozen tissue and sample B for formalinfixed tissue. The protein samples were reduced by DTT and alkylated by iodoacetamide. Then the solutions were diluted to 1 M guanidine-HCl, and the pH values were adjusted to 8.1. Finally, trypsin was added (trypsin/protein, 1:50) and incubated at 37 C for 20 h. (2) For the protocol using 2% SDS with heating, formalin-fixed mouse liver tissue was homogenized in lysis buffer (40 mM Tris and 2% SDS, pH 8.2), and then sonicated for 180 s. Then the mixture was incubated at 100 C for 20 min and 60 C for 2 h. After centrifugation at 25 000g for 1 h, the supernatant was collected as protein sample C. The protein sample was reduced by DTT and alkylated by iodoacetamide. Then the solution was diluted to 0.1% SDS, and the pH was adjusted to 8.1. Finally, trypsin was added (trypsin/ protein, 1:50) and incubated at 37 C for 20 h. The tryptic digest was enrichment with an SCX trap column. (3) For the protocol with direct digestion of tissue homogenate, formalin-fixed mouse liver tissue was homogenized in lysis buffer (40 mM Tris, 6 M guanidine-HCl, and 65 mM DTT, pH 8.2) and then sonicated for 180 s. The proteins in the homogenate were reduced by DTT and alkylated by iodoacetamide. Then, the solution was diluted to 1 M guanidine-HCl, and the pH was adjusted to 8.1. Trypsin was added and incubated at 37 C for 20 h. The resulting tryptic digest was collected after centrifugation. The resulted sample was referred as sample D. (4) For the protocol using 6 M guanidine-HCl with heating, formalinfixed mouse liver tissue was homogenized in lysis buffer (40 mM Tris, 6 M guanidine-HCl, and 65 mM DTT, pH 8.2) and then sonicated for 180 s. The mixture was incubated at 100 C for 30 min. After centrifugation, the supernatant was collected as protein sample E, and the pellet was also collected for further processing. The preparation of tryptic digest of sample E was as described above. (5) For the protocol with CNBr treatment, the pellet from the previous protocol was mixed 90% formic acid and cyanogens bromide as reported.24 Briefly, 90% formic acid was added to the pellet and incubated for 5 min at room temperature. Then CNBr was added to the concentration of 1 g/mL, and the solution was incubated overnight at room temperature in the dark. On the following day, the pH was adjusted to 8.5. The sample F was lyophilized and then was redissolved in the buffer containing 40 mM Tris and 6 M guanidine-HCl. From this point forward, the sample was treated identically to other protocols. The schematic of the protocols was shown in Figure 1.
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specific antigen, prostatic acid phosphatase, and macrophage inhibitory cytokine-1.15 Shotgun proteomics was also applied to the analysis of FFPE cell-block of a human lymphoma cell line; the results were comparable to those obtained with lysates from a fresh specimen of the lymphoma cell line.16 Recently, the extraction of proteins from formalin-fixed tissue was conducted by incubation of these tissue sections in lysis buffer containing 2% SDS at high-temperature.17 Hwang et al.18 developed a methodology termed direct tissue proteomics, and it was also concluded that the extraction condition, lysis buffer containing 2% SDS at high temperature, was a better protocol to extract proteins. Although only a few papers published the shotgun method used to analyze the proteome of formalinfixed tissue, these studies indicated the possibility of using the archival collected formalin-fixed tissues for discovery-driven biomarker research. The most important issue in shotgun proteome analysis of formalin-fixed tissue is to efficiently extract proteins or peptides from the fixed tissues. Except Hood et al., who used commercial Liquid Tissue buffer and no detailed information regarding the composition was given, other researchers mainly used lysis buffer containing 2% SDS to extract proteins from formalinfixed tissue. However, the SDS concentration in protein extract must be reduced before enzymatic digestion because trypsin can only tolerate less than 0.1% SDS.19 The SDS concentration could be reduced simply by diluting the protein extract more than 20-fold. This approach is simple, while some hydrophobic proteins may precipitate because of the low SDS concentration. Most SDS can be removed by dialysis or precipitation, but it is time-consuming and the recovery is relatively low.20 It is also well-known that the presence of SDS in the sample is deleterious to reversed-phase separation. Therefore, it is preferable to develop protocols without utilizing SDS. In this report, several different methods using lysis buffer without SDS were investigated for the preparation of protein samples from formalin-fixed tissues of mouse liver. We found that efficient extraction of proteins from formalin-fixed tissue could be achieved by incubation of the tissue in lysis buffer containing 6 M guanidine-HCl at high temperature. In previous reports, proteins identified from formalin-fixed tissue were classified by Gene ontology, and this found proteins which had a broad range of molecular functions and arose from every cell compartment. But the identified peptides and proteins were not comprehensively characterized. In this study, the peptides and proteins identified from formalin-fixed tissue and frozen fresh tissue were first comprehensively compared in term of some physicochemical properties such as amino acid composition, hydrophobicity, pI, and so on. We found a majority of the resulting peptides were unmodified, and slight difference was observed between the proteins identified from formalinfixed tissue and frozen fresh tissue.

Experimental Section
Materials. Magic C18AQ (5 m, 100 pore) was purchased from Michrom BioResources (Auburn, CA). All the water used in the experiment was purified using a Mill-Q system (Millipore, Bedford, MA). Dithiothreitol (DTT) and iodoacetamide were all purchased from Sino-American Biotechnology Corporation (Beijing, China). TPCK-trypsin, guanidine hydrochloride, and sodium dodecyl sulfate (SDS) were obtained from Sigma (St. Louis, MO). Tris was from Amersco (Solon, OH). Formic acid was obtained from Fluka (Buches, Germany). Acetonitrile (ACN,

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Jiang et al. Table 2. Total Numbers of Peptides and Proteins Detected from Frozen Mouse Liver Tissues and Formalin-Fixed Mouse Liver Tissues
number of identified proteinsa proteins identified with two unique peptides minimumb number of unique peptides

sample

A B C D E F Figure 1. Summary of protocols for protein extraction. Table 1. Protein Concentration of Samples A, B, C, and E (n ) 3)
sample A B C E

976 130 820 331 827 526

480 57 395 106 470 202

3207 352 2540 589 3005 1129

a Accepted fully tryptic peptides only. Xcorr at least 1.9, 2.2, and 3.75 for singly, doubly, and triply charged peptide ions, respectively. Cn g 0.15. False-positive rate less than 5%. b In addition of a, proteins identified with at least two peptides for each protein were accepted.

Protein concentration (mg/mL)

17.7 ( 1.2

0.82 ( 0.13

6.4 ( 0.66

10.9 ( 0.87

HPLC and Mass Spectrometry. Columns were packed using a homemade pneumatic pressure cell at constant nitrogen gas pressure of about 580 psi with a slurry packing method.25 For the preparation of analytical column, one end of a 75 m i.d. fused silica capillary was first manually pulled to a fine point of 5 m with a flame torch. The C18 particles were then packed until the packing section reached the length of 12 cm. The HPLC-MS/MS system consisted of a quaternary Surveyor pump, a Surveyor autosampler, and an LTQ linear ion trap mass spectrometer equipped with a nanospray source (Thermo, San Jose, CA). The two buffer solutions used for the quaternary pump were 0.1% formic acid (mobile-phase A) and 99.9% ACN/ 0.1% formic acid (mobile-phase B). The temperature of the iontransfer capillary was set at 200 C. The spray voltage was set at 1.8 V, and the normalized collision energy was set at 35.0%. An automated gain control function was used to manage the number of ions injected into the ion trap. One microscan was set for each MS and MS/MS scan. All MS and MS/MS spectra were acquired in the data-dependent mode. The mass spectrometer was set so that 1 full MS scan was followed by 10 MS/ MS scans on the 10 most intense ions. The dynamic exclusion function was set as follows: repeat count 2, repeat duration 30 s, and exclusion duration 90 s. System control and data collection were done by Xcalibur software version 1.4 (Thermo). Peptides were eluted using a linear gradient of 5% mobile-phase B to 35% mobile-phase B in 120 min, then 80% mobile-phase B in an additional 15 min, all at a flow rate of about 200 nL/ min. Data Analysis. Each digest was analyzed three times by LCMS/MS, and data analysis was based on the cumulative total proteins identified in three reduplicate analyses. The acquired MS/MS spectra were searched against Mouse International Protein Index (IPI) database (v3.17) using the TurboSEQUEST in the BioWorks 3.2 software suite (Thermo). Reversed sequences were appended to the database for the evaluation of false-positive rate. Cysteine residues were searched as static modification of 57.0215 Da. Peptides were searched using fully tryptic cleavage constraints, and up to two missed cleavages sites were allowed for tryptic digestion. The mass tolerances were 2 Da for parent masses and 1 Da for fragment masses.
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The peptides were considered as positive identification if the Xcorr were higher than 1.9 for singly charged peptide, 2.2 for doubly charged peptide, and 3.75 for triply charged peptides, and Cn cutoff values were g0.15. False-positive rates (FPR) were calculated by using the following equation, FPR ) 2n(rev)/ [n(rev) + n(forw)], where n(forw) and n(rev) are the number of peptides identified in proteins with forward (normal) and reversed sequence, respectively.19,26 False-positive rate less than 5% was obtained for the peptide identifications by using theparameters mentioned above. Proteins were assigned subcelluar localization and molecular functions using GoMiner, a Web-based application based on Gene Ontology (GO) (discover.nci.nih.gov/gominer).27 To improve the confidence of the protein identification, only the proteins identified by two peptides per protein minimum were subjected to GO classification. Grand average of hydropathy (GRAVY) values for each unique protein were calculated according to the method of Kyte and Doolittle.28

Results
Extraction of Proteins with Different Protocols. Efficient extraction of proteins is the most critical step for proteome analysis.29 To investigate if the routine protein preparation protocol could be utilized to extract protein from formalinfixed tissue, the same protocol was applied to the preparation of protein samples for fresh-frozen tissue and formalin-fixed tissue of mouse liver. Both tissues were homogenized in the lysis buffer containing 40 mM Tris, 6 M guanidine-HCl, and 65 mM DTT at 4 C. The protocol was referred as using 6 M guanidine-HCl without heating. After centrifugation, the supernatants were collected, and the protein concentrations were determined by the Bradford assay. As shown in Table 1, protein concentration as high as 17.7 mg/mL was obtained for the protein extract prepared from frozen fresh tissue (sample A), while only 0.82 mg/mL was obtained for formalin-fixed tissue (sample B). The protein yield for the fixed tissue sample was about 20-fold lower than that of frozen tissue sample. The resulting tryptic digests were also subjected to LC-MS/MS analysis. The database search results were listed in Table 2. The analysis of the sample from frozen fresh tissue yielded 3207 unique peptides, which matched 976 unique proteins. While the analysis of the sample from the fixed tissue yielded only 352 unique peptides, which matched 130 unique proteins. For more confident identification, the numbers of proteins identified by at least two peptides were 480 and 57 for the frozen sample and formalin-fixed sample, respectively. These results show that the conventional protocol cannot efficiently extract

Shotgun Proteome Analysis of Formalin-Fixed Tissue

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on utilizing lysis buffer containing 2% SDS with heating.15,18,30 The protocol was also applied to prepare protein extract for formalin-fixed mouse liver tissue in this study. The tissue was boiled in a solution of Tris-HCl containing 2% SDS for 20 min followed by incubation at 60 C for 2 h. The protein concentration of the resulting extract was determined to be 6.4 mg/mL (Table 1), which was lower than that of the protocol utilizing 6 M guanidine-HCl with heating. After LC-MS/MS analysis of the resulting tryptic digest, the identification of 2540 unique peptides corresponding to 820 unique proteins was achieved as shown in Table 2. Among those identified proteins, 395 proteins were identified by at least two peptides. As described above, five different sample preparation protocols were investigated for extraction of proteins or peptides from formalin-fixed tissues. As can be seen from Tables 1 and 2, the protocol using 6 M guanidine-HCl with heating yields the best results, and the protocol using 2% SDS with heating yields the second-best results. But the protocols without heating, that is, the protocol using 6 M guanidine-HCl at low temperature and the protocol with direct digestion of the tissue homogenates by trypsin, yielded relatively poor results. Heating treatment of formalin-fixed tissue was typically applied to retrieve antigen in IHC because of the hydrolysis of cross-links between formalin and protein at high temperature.31 The breakdown of the formalin-induced cross-links at high temperature also explains the reason the protocols with heating are more efficient for the extraction of proteins from formalinfixed tissues. However, efficient protein extraction from formalin-fixed tissue cannot be achieved only by the heating treatment. The buffer containing only 40 mM Tris (pH 8.2) was also utilized to extract protein from such tissues with the same condition as the protocol using 6 M guanidine-HCl with heating or using 2% SDS with heating, and very few proteins were extracted. Therefore, proper lysis buffer and heating conditions are both vitally important for protein extraction from formalinfixed tissue. Characterization of Identified Peptides from Frozen Tissue and Formalin-Fixed Tissue. The process of tissue fixation by formalin, a formaldehyde solution, has been divided into two stages:22,31 the primary reaction is an addition reaction between a primary amine group on the protein molecule and an aldehyde group on formaldehyde, R-NH2 + HCHO f R-NHCH2OH, followed by a secondary condensation reaction, R-NHCH2OH + H2N-CO-R f R-NH-CH2-NH-CO-R + H2O. Therefore, proteins in the fixed tissue are primary cross-linked via the side group of lysine residues. Since proteins undergo both intra- and inter-protein covalent cross-linking, it is reasonable that the extraction of these proteins at low temperature is not efficient. As we demonstrated, the proteins in the formalin-fixed tissue could be efficiently extracted with heating due to the breakdown of some cross-links. Trypsin digestion of the resulting proteins will produce three kinds of peptides: peptides without modifications, peptides with some amino acid residues modified, and peptides that are crosslinked to other peptides or form internal cross-links. The crosslinked peptides cannot be identified by the Sequest database search algorithm. The identification of unmodified peptides can be easily achieved by Sequest database searching with standard parameters, while the identification of the modified peptides could only be achieved if the chemistry of the modification is clear. In accordance with the above-mentioned chemical reactions, the primary amine of lysine is modified by CH2OH; therefore, a variable modification with 30 Da was set for the
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protein from formalin-fixed tissue. Since proteins undergo both intra- and inter-protein covalent cross-linking resulting from formalin fixation, it is reasonable to assume that they exist as extensive cross-linked protein networks leading to the difficulties in their efficient extraction. On the basis of these results, proteins in the formalin-fixed tissue are difficult to extract by the conventional method because they are trapped in the network of cross-linked proteins. On the other hand, the cross-linked protein network may be disrupted by trypsin digestion. Therefore, formalinfixed tissue homogenates were directly digested with trypsin to improve the result of protein identification. After incubation with trypsin for 20 h at 37 C, the tissue homogenate was subjected to centrifugation, and the supernatant was collected for LC-MS/MS analysis. After database searching, 589 unique peptides were matched with MS/MS spectra, which resulted in the identification of 331 unique proteins. Among the 331 unique proteins, 106 proteins were matched by two or more peptides (Table 2). Compared with the conventional protocol where only 57 proteins that matched two peptide minimum were identified from the formalin-fixed tissue, this protocol was more effective. But it is far from ideal compared with the number of proteins identified from the frozen fresh tissue. To further improve the efficiency of protein extraction from formalin-fixed tissue and considering the cross-linking of proteins in these tissues, the violent conditions, such as heating, should be tried to increase the recovery of proteins. Thus, the heating treatment was applied to the protocol with 6 M guanidine-HCl. After the formalin-fixed mouse liver tissue was homogenized in lysis buffer (40 mM Tris, 6 M guanidine-HCl, and 65 mM DTT), the resulting mixture was incubated at 100 C for 30 min. The protein concentration was determined to be 10.9 mg/mL, which was comparable with that from frozen fresh tissue. After LC-MS/MS analysis of the resulting tryptic digest, 3005 unique peptides and 827 unique proteins were identified. The number of proteins matched with at least two peptides was 470, which was slightly lower than that for freshfrozen tissue. These results indicated that the protocol using 6 M guanidine-HCl with heating was very efficient for protein extraction from formalin-fixed tissue. The results obtained from formalin-fixed tissue were comparable to those obtained from the frozen fresh tissue. Furthermore, due to the cross linking of proteins by formalin leading to the insolubility of some proteins, there may be some cross-linked proteins that still remained in the pellet. To probe the tissue proteome in depth, the pellet was also processed for LC-MS/MS analysis. Besides the highly cross-linked protein complex, the hydrophobic membrane proteins were also presented in the pellet. It was reported that the membrane proteins could be cleaved off by cyanogens bromide (CNBr), and the identification of membrane proteins could be achieved by LC-MS/MS analysis.24 Similarly, the unlinked portions of the insoluble highly cross-linked complex may also be cleaved off by CNBr for shotgun proteome analysis. Therefore, the pellets were treated with CNBr as the procedure described in the Experimental Section. The resulting fragments from these insoluble proteins were further digested by trypsin for LC-MS/ MS analysis. After database searching, 1129 unique peptides were matched to result in the identification of 526 unique proteins. Among those proteins, 202 proteins were identified by two peptides minimum (Table 2). Previous reported studies of proteins extraction from formalin-fixed tissues for shotgun proteomics was mainly based

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lysine for Sequest search of the MS/MS data obtained from formalin-fixed tissue. Because trypsin cannot cleave peptides with the modified lysine residue, the percentage of tryptic peptides with lysyl termini must be dramatically decreased for formalin-fixed tissue if a majority of lysine residues were modified. The search results showed that only about 5.0% of the identified peptides containing modified lysyl residues and a majority of lysine residues were still unmodified for the proteins obtained from formalin-fixed tissue. Although other modifications may also occur to the lysine residue and other amino acid residues, because the process of formalin fixation and the influence of heating treatment are not well-understood, and the chemistry of modified peptides was not clear, no modification was set for the database search for the characterization of identified peptides/proteins from frozen tissue and formalin-fixed tissue in this study. An important issue when using shotgun proteomics for analysis of formalin-fixed tissue sample is whether the covalent modifications resulting from formalin fixation affect the proteome analysis results. Therefore, the results obtained from the analysis of formalin-fixed tissue and frozen fresh tissue should be compared. In this study, the proteins in frozen fresh tissue were extracted using buffer containing 6 M guanidine-HCl without heating. As we described above, the extraction of proteins from formalin-fixed tissue was also efficient when the buffer containing 6 M guanidine-HCl was used in combination with heating treatment. Table 2 shows the numbers of identified peptides and proteins were comparable for these two cases. Because the same buffer was utilized and a similar number of proteins and peptides identified, the comparison for the proteome analysis results on the peptide and protein levels was based on the two datasets mentioned above (Sample A vs Sample E in Table 2; for a complete lists, see Supporting Information data 1 and 2). And 55% of the identified proteins (281 proteins) were observed in both samples (for complete list, see Supporting Information data 3). Besides the modifications to lysine residues, the modification of other residues may also happen during the formalin fixation. Furthermore, the heating treatment for extraction of proteins may lead to the occurrence of additional modifications. Therefore, it is necessary to evaluate the influence of these modifications on the shotgun proteome analysis of formalin-fixed tissue. If the modification of a certain residue did happen in the formalin-fixed tissue sample, the number of unmodified peptides containing the residue must decrease, and thus, the percentage of the residue among all the identified peptides should also decrease. The total numbers of amino acid residues in those identified unique peptides from the frozen tissue sample and the formalin-fixed tissue sample were 24 310 and 23 738, respectively. The percentages of the number of individual amino acid residues among the total number of amino acid residues in the identified unmodified peptides from frozen tissue and fixed tissue were shown in Figure 2. The percentages of lysine residues are 6.33% and 5.73% for frozen sample and formalin-fixed sample, respectively. The percentage of lysine residues for the formalin-fixed sample is about 10% lower than that for the frozen sample, which indicated that a portion of lysine residues was modified during formalin fixation. This is consistent with the results derived from the decreased percentage of peptides with lysyl termini. Besides that of lysine residue, the percentages of phenylalanine (F), asparagine (N), tryptophan (W), and tyrosine (Y) residues in the peptides identified from fixed tissue sample were obviously lower than those from
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Figure 2. Distribution of amino acid residues in the peptides identified from frozen mouse liver tissue and formalin-fixed mouse liver tissue.

frozen tissue sample, which indicated that the number of unmodified peptides containing these residues decreased in the fixed tissue sample. The decrease of lysine residue percentage is largely because due to formalin modifying this residue. Since the other residues do not likely react with formaldehyde, the decreasing of their percentages may be caused by the heating treatment, which results in the degradation of these residues. Figure 2 shows that all the amino acid residues were observed in the formalin-fixed tissue sample and the percentages of these residues were quite similar to those of frozen tissue. Except the rare amino acid residue, tryptophan, which account for only about 1% in protein sequence, the decrease of the percentages of other residues were not significant. This means that a majority of peptides obtained from formalin-fixed tissue were unmodified. This is the reason that shotgun proteome analysis approach could be successfully applied to analysis of formalin-fixed tissues. Characterization of Identified Proteins from Frozen Tissue and Formalin-Fixed Tissue. Both the formalin fixation process and the heating treatment for sample preparation will lead to the modifications of side chains of some proteins, and might even induce the degradation of some proteins. An important issue for proteome analysis of formalin-fixed tissue is whether the integrity of the proteome is compromised by these modifications. To answer this question, the identified proteins from frozen fresh liver tissue and those from formalin-fixed tissue should be comprehensively compared. As referred above, the amino acid composition of peptides identified from formalin-fixed tissue was slightly different from that of peptides identified from frozen tissue because of amino acid modifications induced by formalin reaction and heating treatment. However, at the protein level, do such modifications affect proteins identified from formalin-fixed tissue in contrast with frozen tissue? To answer this question, the amino acid composition of proteins identified from frozen tissue and formalin-fixed tissue was investigated in detail. We found that the distributions of the average amino acid composition of proteins identified from frozen fresh tissue and formalin-fixed tissue, as shown in Figure 3, were very similar. Slight discrepancies were observed for some amino acids. For example, proteins with higher percentage of A, K, and R residues were observed for the formalin-fixed sample, while proteins with higher percentage of D, F, and Y residues were observed for the frozen tissue. Interestingly, the discrepancies in the amino

Shotgun Proteome Analysis of Formalin-Fixed Tissue

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Figure 3. Distribution of average amino acid composition for the proteins identified from frozen mouse liver tissue and formalinfixed mouse liver tissue.

Figure 4. Distribution of Grand average of hydropathy (GRAVY) of proteins identified from frozen mouse liver tissue and formalinfixed liver tissue.

acid composition were different between the protein level and peptide level. As we described above, less peptides containing lysine residue (K) were identified in formalin-fixed tissue because of the modifications induced by the formalin fixation. But at the protein level, proteins with a higher percentage of lysine residue were identified in formalin-fixed tissue. This is surprising, considering the proteins with higher percentage of K residues are more heavily cross-linked and. therefore. these proteins should not be easily extracted nor accessible for trypsin. But the opposite results were observed in this study. This probably can be explained by the fact that the most of the cross-links were opened by the heating treatment during the protein extraction step. Overall, the profiles for the distributions of proteins with different amino acid composition were almost identical, which indicates the modifications of some amino acid residues induced by the formalin fixation hardly impact the integrity of the proteome for the analysis of fixed tissue. As addressed above, proteins with relative higher percentage of K and R residues were identified from formalin-fixed tissue. Since the K and R residues are very hydrophilic, the proteins identified from formalin-fixed tissue probably are more hydrophilic than those identified from frozen tissue. To compare the hydrophobicity of the identified proteins, the grand average hydrophobicity (GRAVY) values for all proteins identified from the fixed tissue and frozen tissue were determined according to Kyte and Doolittle.28 The higher the GRAVY value is, the more hydrophobic the protein is. The results are shown in Figure 4. More proteins were identified from fixed tissue in the range of GRAVY value < -0.5, which indicated that the more hydrophilic proteins were identified from formalin-fixed tissue. The average GRAVY values for the proteins identified from the fixed tissue and frozen tissue were -0.333 and -0.287, respectively. These results indicate that shotgun proteome analysis of formalinfixed tissue is slightly bias toward hydrophilic proteins. The distributions of isoelectric point (pI) and molecular weight (MW), which were also important physicochemical properties of proteins, were examined. Proteins identified from frozen tissue and formalin-fixed tissue all spread in a broad range of pI and MW as shown in Figure 5. However, there was some difference in specific scope of pI and MW. The percentage of proteins identified from formalin-fixed tissue is higher than that from frozen tissue in the scope of 4-6 and >10 of pI values. And in the range of 6-10 of pI values, the percentage of

proteins identified from formalin-fixed tissue is lower than that from frozen tissue. The average pI values for the proteins identified from formalin-fixed tissue and frozen tissue were 7.44 and 7.26, respectively. More basic proteins were identified from formalin-fixed tissue. This is consistent with the distribution of identified protein with different amino acid composition as shown in Figure 3, where more proteins with high percentage of basic amino acid residues, K and R, were identified from the formalin-fixed tissue. For the difference in the distribution of MW as shown in Figure 5, more proteins were identified from formalin-fixed tissue in the range of 15-30 kDa. The average MW of the proteins identified from formalin-fixed and frozen tissue was 55.8 and 58.9 kDa, respectively. The proteins identified from the formalin-fixed tissue are slightly biased toward low MW. The proteins identified in the analysis of fresh frozen tissue and formalin-fixed tissue were subjected to classification using Gene Ontology. The resulting cellular localization of proteins was shown in Figure 6. Similar to those identified from fresh frozen tissue, the proteins identified from the formalin-fixed tissue also came from different cell compartments. Slightly less membrane proteins were identified from formalin-fixed tissue. The proteins identified from the formalin-fixed tissue also have a broad range of molecular functions as shown in Figure 6. Compared with frozen tissue, more proteins with molecular structural function were identified from formalin-fixed tissue. Although a slight bias was observed, broad protein coverage could be obtained by analysis of formalin-fixed tissue. Proteome samples are typically very complex, and mass spectrometer cannot collect tandem mass spectra from all eluting peptides because of limited scan rate. The proteins identified by the shotgun proteomics approach with different LC-MS/ MS runs are not exactly the same even for the same sample. Liu et al.32 compared the proteins identified from 9 replicate SCX-RPLC-MS/MS runs for a same yeast protein digest sample and found that about 35.4% were identified in every run. However, in this study two different proteome samples, one from formalin-fixed tissue and another from frozen tissue, were analyzed, and it was found that more than 55% of the identified proteins (based on two peptides or more for each protein) were the same. Therefore, the proteome analysis results obtained from formalin-fixed tissue were quite comparable to those obtained from frozen tissue. The fact that proteome coverage obtained from formalin-fixed tissue sample by shotgun proJournal of Proteome Research Vol. 6, No. 3, 2007 1043

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Jiang et al.

Figure 5. Distribution of molecular weight (MW) and pI of proteins identified from frozen mouse liver tissue and formalin-fixed mouse liver tissue. (A) MW distribution with 5 kDa increments; (B) pI distribution of the proteins with 0.5 pH unit increments.

teome analysis was not compromised because of formalin fixation indicated that the archived fixed tissues might be good samples for biomarker discovery instead of frozen-fresh tissue.

Discussion
Fresh frozen tissue is difficult to obtain in large numbers, whereas extensive archives of animal and human well-defined formalin-fixed tissue which contains associated experimental and clinical information representing a highly valuable reservoir of potential biomarkers are readily available. Unfortunately, effective and routine analysis of proteins in formalin-fixed tissue has been limited to IHC. This limitation is due to the chemical cross-linking properties of the common fixative reagent of formalin. The proteins extracted from the formalinfixed tissue have different physicochemical properties from those of fresh proteins. For example, the pI values of proteins will change because of the decrease of primary amines resulting from the reaction of lysine side chains with formaldehyde; the MW of the extracted proteins may also increase because of the possible cross-link with other proteins and biomolecules. Moreover, the reactions between protein and formaldehyde are
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not likely gone completely, that means a series of products may result for a single protein. Because of above reasons, the conventional protein analysis approaches are not suitable for the analysis of proteins extracted from formalin-fixed tissue. Because of the poor results obtained from 2D-PAGE resolution of proteins extracted from formalin-fixed tissue, Ahram et al.33 concluded that the utility of formalin-fixed tissue for protein separation studies was limited. Because of the development of the shotgun proteomics strategy in recent years, it has become possible to conduct discovery-driven investigation in formalin-fixed tissue.10 The separation takes place at the protein level in conventional protein analysis approaches, while the separation takes place at the peptide level in shotgun proteomics. The proteins are digested into peptides, and then these peptides are analyzed by LC-MS/MS in shotgun proteomics. Although some side chains of protein are modified by formaldehyde in the formalinfixed tissue, many unmodified peptides are generated after trypsin digestion. The identification of proteins can be easily achieved by LC-MS/MS analysis of the unmodified peptides. In this study, all 20 amino acid residues were found in the

Shotgun Proteome Analysis of Formalin-Fixed Tissue

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quite similar. The difference in the amino acid composition of proteins identified from the two samples was first investigated in this study, and we found the distributions were also quite similar. Because relatively more proteins with high percentage of K and R residues were observed in formalin-fixed tissue, the proteins identified from formalin-fixed tissue were slightly more hydrophilic and basic. A slight bias toward low MW proteins for shotgun proteome analysis of fixed tissue was also observed. But for all the properties of protein investigated, including amino acid composition, hydrophobicity, pI value, and MW, the overall distribution was all similar, which indicated that the formalin fixation dose not adversely affect the diversity of proteins identified from tissue sample. The analysis of protein extract prepared by the protocol of 6 M guanidine-HCl with heating for formalin-fixed tissue resulted in the identification of 470 unique proteins based on two or more peptides for each protein. The number of identified proteins was comparable to that identified from frozen-fresh tissue, and 55% of identified proteins were observed in both samples. All of these results indicated that similar proteome coverage could be obtained by shotgun proteome analysis of formalin-fixed tissue. Moreover, notably, the incomplete overlap of the proteins identified from both samples was observed. As previous reports described,11,34,35 in any given large-scale proteomic experiment, only a subset of the entire proteome was identified due to technical limitations of current proteomic technologies, and shotgun proteome approaches might expect to identify different peptide subsets from identical biological samples, even across replicate analyses. This was a main reason that peptides/ proteins identified from frozen tissue and formalin-fixed tissue were not exactly the same. To comprehensively analyze the proteome of formalin-fixed tissue, more effective LC-MS technologies and the variety of fractionation approaches should be applied in routine implementation of shotgun proteomic profiling platform. Furthermore, because of the importance of post-translational modifications, such as phosphorylation and glycosylation, further studies should be performed to investigate if the actual form of the protein post-modifications were preserved in formalin-fixed tissue. As valuable proteome information can be retrieved from formalin-fixed tissue samples, the shotgun proteome analysis approach provides the ability to unlock the proteome of the worlds vast reservoir of archived tissue for large-scale discovery and validation of biomarkers to improve disease diagnosis and therapy. As we know, the archived formalin-fixed tissue were typically collected at different place and time. Before the formalin-fixed tissue could be used as an alternative to frozenfresh tissue for biomarker discovery, studies examining formalin-fixed tissues that have been stored for varying lengths of time and collected from different labs should be conducted to determine if these conditions affect proteome analysis results.

Figure 6. Gene ontology cellular location and molecular function of the identified proteins from frozen mouse liver tissue and formalin-fixed mouse liver tissue. (A) Cellular location; (B) molecular function.

peptides identified from formalin-fixed tissue, and the distribution of these amino acid residues was very similar to that of frozen tissue. These results indicated that a majority of peptides from formalin-fixed tissue were unmodified. This is the reason shotgun proteomics approach could be successfully applied to the analysis of formalin-fixed tissue. Recently, increased attention was paid to shotgun proteome analysis of formalin-fixed tissue.10,15-18,30 Most of these studies use SDS lysis buffer to extract proteins from formalin-fixed tissue. We found in this study that proteins can be more efficiently extracted from the fixed tissue using 6 M guanidineHCl with heating. Considering highly cross-linked protein complex and hydrophobic membrane proteins still presented in the pellet, the remaining pellet was further treated with CNBr. Although the protocol with CNBr treatment resulted in a relatively low number of protein identifications, it generated about 17% of membrane proteins which was a much higher number than that obtained using the 6 M guanidine-HCl protocol. To comprehensively analyze the proteome of formalin-fixed tissue, the use of multiple sample preparation protocols together is preferable. Combining the proteins identified through the three protocols, that is, 6 M guanidine-HCl with heating, 2% SDS with heating, and CNBr treatment, resulted in the identification of 772 unique proteins with two peptides minimum for each protein. The proteins identified in the formalin-fixed tissue/cell and frozen-fresh tissue/cell were also classified by Gene Ontology in previous reports.15-17 Similarly to this study, a slight bias was observed, but the overall profiles of the protein distributions in term of molecular function and cellular localization were

Conclusion
The development of methods to use state-of-the-art proteomic discovery tools to analyze formalin-fixed tissue provides an exciting new opportunity to identify disease-specific biomarkers in pathologically defined samples. In this study, protocols for the efficient extraction of proteins from formalin-fixed tissue were developed, which enabled the use of highthroughput shotgun proteome analysis approach for large-scale and in-depth analysis of proteins presented in archived tissue
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samples. The peptides and proteins identified from formalinfixed tissue were comprehensively characterized. It was found that the formalin fixation does not compromise the proteome coverage of the analysis.

Jiang et al.
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Acknowledgment. Financial supports from the National Natural Sciences Foundation of China (No. 20327002), the China State Key Basic Research Program Grant (2005CB522701), and the Knowledge Innovation program of DICP to H.Z. are gratefully acknowledged. Supporting Information Available: Tables listing the complete datasets for the comparison of the proteome analysis results on the peptide and protein levels, and the dataset of the identified proteins observed in both samples. This material is available free of charge via the Internet at http://pubs.acs.org. References
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