2‐D PAGE and MS analysis of proteins from formalin‐fixed,paraffin‐embedded tissues

In the past decade, encouraging results have been obtained in extraction and analysis of proteins from formalin‐fixed, paraffin‐embedded (FFPE) tissues. However, 2‐D PAGE protein maps with satisfactory proteomic information and comparability to fresh tissues have never been described to date. In the present study, we report 2‐D PAGE separation and MS identification of full‐length proteins extracted from FFPE skeletal muscle tissue. The 2‐D protein profiles obtained from FFPE tissues could be matched to those achieved from frozen tissues replicates. Up to 250 spots were clearly detected in 2‐D maps of proteins from FFPE tissue following standard mass‐compatible silver staining. Protein spots from both FFPE and frozen tissue 2‐D gels were excised, subjected to in situ hydrolysis, and identified by MS analysis. Matched spots produced matched protein identifications. Moreover, 2‐D protein maps from FFPE tissues were successfully subjected to Western immunoblotting, producing comparable results to fresh‐frozen tissues. In conclusion, this study provides evidence that, when adequately extracted, full‐length proteins from FFPE tissues might be suitable to 2‐D PAGE‐MS analysis, allowing differential proteomic studies on the vast existing archives of healthy and pathological‐fixed tissues.     

Impact of fixation time on GeLC-MS/MS proteomic profiling of formalin-fixed, paraffin-embedded tissues

Formalin-fixed, paraffin-embedded (FFPE) tissue banks represent an invaluable resource for biomarker discovery. Recently, the combination of full-length protein extraction, GeLC-MS/MS analysis, and spectral counting quantification has been successfully applied to mine proteomic information from these tissues. However, several sources of variability affect these samples; among these, the duration of the fixation process is one of the most important and most easily controllable ones. To assess its influence on quality of GeLC-MS/MS data, the impact of fixation time on efficiency of full-length protein extraction efficiency and on quality of label-free quantitative data was evaluated. As a result, although proteins were successfully extracted from FFPE liver samples fixed for up to eight days, fixation time appeared to negatively influence both protein extraction yield and GeLC-MS/MS quantitative proteomic data. Particularly, MS identification efficiency decreased with increasing fixation times. Moreover, amino acid modifications putatively induced by formaldehyde were detected and characterized. These results demonstrate that proteomic information can be achieved also from tissue samples fixed for relatively long times, but suggest that variations in fixation time need to be carefully taken into account when performing proteomic biomarker discovery studies on fixed tissue archives.     

Application of 2-D DIGE to formalin-fixed, paraffin-embedded tissues

The ability to investigate the proteome of formalin-fixed, paraffin-embedded (FFPE) tissues can be considered a major recent achievement in the field of clinical proteomics. However, gel-based approaches to the investigation of FFPE tissue proteomes have lagged behind, mainly because of insufficient quality of full-length protein extracts. Here, the 2-D DIGE technology was investigated for applicability to FFPE proteins, for internal reproducibility among replicate FFPE extracts, and for comparability between FFPE and fresh-frozen tissue profiles. The 2-D DIGE patterns obtained upon labeling and electrophoresis of replicate FFPE tissue extracts were highly reproducible, with satisfactory resolution and complexity. Moreover, the implementation of DIGE enabled to highlight and characterize the consistent differences found in the FFPE profiles compared with fresh-frozen profiles, represented by an acidic shift, directly correlated to the protein pI value, and by a reduction in spot signal intensity, directly correlated to molecular weight and percentage of lysine residues. Being constantly and reproducibly present in all FFPE tissue extract replicates at similar extents, these modifications do not appear to hinder the comparative analysis of FFPE tissue extracts by 2-D DIGE, opening the way to its application for the differential proteomic investigation of archival tissue repositories.     

Critical comparison of sample preparation strategies for shotgun proteomic analysis of formalin-fixed,paraffin-embedded samples: insights from liver tissue

Background

The growing field of formalin-fixed paraffin-embedded (FFPE) tissue proteomics holds promise for improving translational research. Direct tissue trypsinization (DT) and protein extraction followed by in solution digestion (ISD) or filter-aided sample preparation (FASP) are the most common workflows for shotgun analysis of FFPE samples, but a critical comparison of the different methods is currently lacking.

Experimental design

DT, FASP and ISD workflows were compared by subjecting to the same label-free quantitative approach three independent technical replicates of each method applied to FFPE liver tissue. Data were evaluated in terms of method reproducibility and protein/peptide distribution according to localization, MW, pI and hydrophobicity.

Results

DT showed lower reproducibility, good preservation of high-MW proteins, a general bias towards hydrophilic and acidic proteins, much lower keratin contamination, as well as higher abundance of non-tryptic peptides. Conversely, FASP and ISD proteomes were depleted in high-MW proteins and enriched in hydrophobic and membrane proteins; FASP provided higher identification yields, while ISD exhibited higher reproducibility.

Conclusions

These results highlight that diverse sample preparation strategies provide significantly different proteomic information, and present typical biases that should be taken into account when dealing with FFPE samples. When a sufficient amount of tissue is available, the complementary use of different methods is suggested to increase proteome coverage and depth.     

Integrated and convenient procedure for protein extraction from formalin‐fixed,paraffin‐embedded tissues for LC‐MS/MS analysis

Because fresh‐frozen tissue samples associated with long‐term clinical data and of rare diseases are often unobtainable at the present time, formalin‐fixed paraffin‐embedded (FFPE) tissue samples are considered a highly valuable resource for researchers. However, protein extraction from FFPE tissues faces challenges of deparaffinization and cross‐link reversion. Current procedures for protein extraction from FFPE tissue require separate steps and toxic solvents, resulting in inconvenience in protein extraction. To overcome these limitations, an integrated method was developed using nontoxic solvents in four types of FFPE tissues. The average amount of proteins from three replicates of bladder, kidney, liver, and lung FFPE tissues were 442.6, 728.9, 736.4, and 694.7 μg with CVs of 7.5, 5.8, 2.4, and 4.5%, respectively. Proteomic analysis showed that 348, 417, 607, and 304 unique proteins were identified and quantified without specification of isoform by a least two peptides from bladder, kidney, liver, and lung FFPE tissue samples, respectively. The analysis of individual protein CV demonstrated that 97–99% of the proteins were quantified with a CV ≤ 30%, verifying the reproducibility of the integrated protein extraction method. In summary, the developed method is high‐yield, reproducible, convenient, simple, low cost, nonvolatile, nonflammable, and nontoxic.     

Unrestricted modification search reveals lysine methylation as major modification induced by tissue formalin fixation and paraffin embedding

Formalin‐fixed paraffin‐embedded (FFPE) tissue is considered as an appropriate alternative to frozen/fresh tissue for proteomic analysis. Here we study formalin‐induced alternations on a proteome‐wide level. We compared LC‐MS/MS data of FFPE and frozen human kidney tissues by two methods. First, clustering analysis revealed that the biological variation is higher than the variation introduced by the two sample processing techniques and clusters formed in accordance with the biological tissue origin and not with the sample preservation method. Second, we combined open modification search and spectral counting to find modifications that are more abundant in FFPE samples compared to frozen samples. This analysis revealed lysine methylation (+14 Da) as the most frequent modification induced by FFPE preservation. We also detected a slight increase in methylene (+12 Da) and methylol (+30 Da) adducts as well as a putative modification of +58 Da, but they contribute less to the overall modification count. Subsequent SEQUEST analysis and X!Tandem searches of different datasets confirmed these trends. However, the modifications due to FFPE sample processing are a minor disturbance affecting 2–6% of all peptide‐spectrum matches and the peptides lists identified in FFPE and frozen tissues are still highly similar.     

Tissue proteomics using chemical immobilization and mass spectrometry

Proteomics analysis is important for characterizing tissues to gain biological and pathological insights, which could lead to the identification of disease-associated proteins for disease diagnostics or targeted therapy. However, tissues are commonly embedded in optimal cutting temperature medium (OCT) or are formalin-fixed and paraffin-embedded (FFPE) in order to maintain tissue morphology for histology evaluation. Although several tissue proteomic analyses have been performed on FFPE tissues using advanced mass spectrometry (MS) technologies, high-throughput proteomic analysis of OCT-embedded tissues has been difficult due to the interference of OCT in the MS analysis. In addition, molecules other than proteins present in tissues further complicate tissue proteomic analysis. Here, we report the development of a method using chemical immobilization of proteins for peptide extraction (CIPPE). In this method, proteins are chemically immobilized onto a solid support; interferences from tissues and OCT embedding are removed by extensive washing of proteins conjugated on the solid support. Peptides are then released from the solid phase by proteolysis, enabling MS analysis. This method was first validated by eliminating OCT interference from a standard protein, human serum albumin, where all of the unique peaks contributed by OCT contamination were eradicated. Finally, this method was applied for the proteomic analysis of frozen and OCT-embedded tissues using iTRAQ (isobaric tag for relative and absolute quantitation) labeling and two-dimensional liquid chromatography tandem mass spectrometry. The data showed reproducible extraction and quantitation of 10,284 proteins from 3996 protein groups and a minimal impact of OCT embedding on the analysis of the global proteome of the stored tissue samples.     

Validation of a robust proteomic analysis carried out on formalin‐fixed paraffin‐embedded tissues of the pancreas obtained from mouse and human

A number of reports have recently emerged with focus on extraction of proteins from formalin‐fixed paraffin‐embedded (FFPE) tissues for MS analysis; however, reproducibility and robustness as compared to flash frozen controls is generally overlooked. The goal of this study was to identify and validate a practical and highly robust approach for the proteomics analysis of FFPE tissues. FFPE and matched frozen pancreatic tissues obtained from mice (n = 8) were analyzed using 1D‐nanoLC‐MS(MS)² following work up with commercially available kits. The chosen approach for FFPE tissues was found to be highly comparable to that of frozen. In addition, the total number of unique peptides identified between the two groups was highly similar, with 958 identified for FFPE and 1070 identified for frozen, with protein identifications that corresponded by approximately 80%. This approach was then applied to archived human FFPE pancreatic cancer specimens (n = 11) as compared to uninvolved tissues (n = 8), where 47 potential pancreatic ductal adenocarcinoma markers were identified as significantly increased, of which 28 were previously reported. Further, these proteins share strongly overlapping pathway associations to pancreatic cancer that include estrogen receptor α. Together, these data support the validation of an approach for the proteomic analysis of FFPE tissues that is straightforward and highly robust, which can also be effectively applied toward translational studies of disease.     

Temporal proteomic analysis of IGF‐1R signalling in MCF‐7 breast adenocarcinoma cells

Dysregulation of the insulin‐like growth factor 1 receptor signalling network is implicated in tumour growth and resistance to chemotherapy. We explored proteomic changes resulting from insulin‐like growth factor 1 stimulation of MCF‐7 adenocarcinoma cells as a function of time. Quantitative analysis using iTRAQ? reagents and 2‐D LC‐MS/MS analysis of three biological replicates resulted in the identification of 899 proteins (p≤0.05) with an estimated mean false‐positive rate of 2.6%. Quantitative protein expression was obtained from 681 proteins. Further analysis by supervised k‐means clustering identified five temporal clusters, which were submitted to the FuncAssociate server to assign overrepresented gene ontology terms. Proteins associated with vesicle transport were significantly overrepresented. We further analyzed our data set for proteins showing temporal significance using the software, extraction and analysis of differential gene expression, resulting in 20 significantly and temporally changing proteins (p≤0.1). These significant proteins play roles in, among others, altered glucose metabolism (lactate dehydrogenase A and pyruvate kinase M1/M2) and cellular stress (nascent polypeptide‐associated complex subunit α and heat shock (HSC70) proteins). We used multiple reaction monitoring to validate these interesting proteins and have revealed several differences in relative peptide expression corresponding to protein isoforms and variants.     

Generation of high‐quality protein extracts from formalin‐fixed,paraffin‐embedded tissues

A wealth of information on proteins involved in many aspects of disease is encased within formalin‐fixed paraffin‐embedded (FFPE) tissue repositories stored in hospitals worldwide. Recently, access to this “hidden treasure” is being actively pursued by the application of two main extraction strategies: digestion of the entangled protein matrix with generation of tryptic peptides, or decrosslinking and extraction of full‐length proteins. Here, we describe an optimised method for extraction of full‐length proteins from FFPE tissues. This method builds on the classical “antigen retrieval” technique used for immunohistochemistry, and allows generation of protein extracts with elevated and reproducible yields. In model animal tissues, average yields of 16.3 μg and 86.8 μg of proteins were obtained per 80 mm² tissue slice of formalin‐fixed paraffin‐embedded skeletal muscle and liver, respectively. Protein extracts generated with this method can be used for the reproducible investigation of the proteome with a wide array of techniques. The results obtained by SDS‐PAGE, western immunoblotting, protein arrays, ELISA, and, most importantly, nanoHPLC‐nanoESI‐Q‐TOF MS of FFPE proteins resolved by SDS‐PAGE, are presented and discussed. An evaluation of the extent of modifications introduced on proteins by formalin fixation and crosslink reversal, and their impact on quality of MS results, is also reported.     

Protein Extraction of Formalin-fixed, Paraffin-embedded Tissue Enables Robust Proteomic Profiles by Mass Spectrometry

Global mass spectrometry (MS) profiling and spectral count quantitation are used to identify unique or differentially expressed proteins and can help identify potential biomarkers. MS has rarely been conducted in retrospective studies, because historically, available samples for protein analyses were limited to formalin-fixed, paraffin-embedded (FFPE) archived tissue specimens. Reliable methods for obtaining proteomic profiles from FFPE samples are needed. Proteomic analysis of these samples has been confounded by formalin-induced protein cross-linking. The performance of extracted proteins in a liquid chromatography tandem MS format from FFPE samples and extracts from whole and laser capture microdissected (LCM) FFPE and frozen/optimal cutting temperature (OCT)–embedded matched control rat liver samples were compared. Extracts from FFPE and frozen/OCT–embedded livers from atorvastatin-treated rats were further compared to assess the performance of FFPE samples in identifying atorvastatin-regulated proteins. Comparable molecular mass representation was found in extracts from FFPE and OCT-frozen tissue sections, whereas protein yields were slightly less for the FFPE sample. The numbers of shared proteins identified indicated that robust proteomic representation from FFPE tissue and LCM did not negatively affect the number of identified proteins from either OCT-frozen or FFPE samples. Subcellular representation in FFPE samples was similar to OCT-frozen, with predominantly cytoplasmic proteins identified. Biologically relevant protein changes were detected in atorvastatin-treated FFPE liver samples, and selected atorvastatin-related proteins identified by MS were confirmed by Western blot analysis. These findings demonstrate that formalin fixation, paraffin processing, and LCM do not negatively impact protein quality and quantity as determined by MS and that FFPE samples are amenable to global proteomic analysis. (J Histochem Cytochem 57:849–860, 2009)     

Pre‐fractionation of rat liver cytosol proteins prior to mass spectrometry‐based proteomic analysis using tandem biomimetic affinity chromatography

Efficient and high resolution separation of the protein mixture prior to trypsin digestion and mass spectrometry (MS) analysis is generally used to reduce the complexity of samples, an approach that highly increases the probability of detecting low‐copy‐number proteins. Our laboratory has constructed an affinity ligand library composed of thousands of ligands with different protein absorbance effects. Structural differences between these ligands result in different non‐bonded protein–ligand interactions, thus each ligand exhibits a specific affinity to some protein groups. In this work, we first selected out several synthetic affinity ligands showing large band distribution differences in proteins absorbance profiles, and a tandem composition of these affinity ligands was used to distribute complex rat liver cytosol into simple subgroups. Ultimately, all the fractions collected from tandem affinity pre‐fractionation were digested and then analyzed by LC‐MS/MS, which resulted in high confidence identification of 665 unique rat protein groups, 1.8 times as many proteins as were detected in the un‐fractionated sample (371 protein groups). Of these, 375 new proteins were identified in tandem fractions, and most of the proteins identified in un‐fractionated sample (290, 80%) also emerged in tandem fractions. Most importantly, 430 unique proteins (64.7%) only characterized in specific fractions, indicating that the crude tissue extract was well distributed by tandem affinity fractionation. All detected proteins were bioinformatically annotated according to their physicochemical characteristics (such as MW, pI, GRAVY value, TM Helices). This approach highlighted the sensitivity of this method to a wide variety of protein classes. Combined usage of tandem affinity pre‐fractionation with MS‐based proteomic analysis is simple, low‐cost, and effective, providing the prospect of broad application in proteomics. Copyright © 2009 John Wiley & Sons, Ltd.     

Proteomic analysis of laser-microdissected paraffin-embedded tissues: (1) Stage-related protein candidates upon non-metastatic lung adenocarcinoma

We used formalin-fixed paraffin-embedded (FFPE) materials for biomarker discovery in cases of lung cancer using proteomic analysis. We conducted a retrospective global proteomic study in order to characterize protein expression reflecting clinical stages of individual patients with stage I lung adenocarcinoma without lymph node involvement (n = 7). In addition, we studied more advanced stage IIIA with spread to lymph nodes (n = 6), because the degree of lymph node involvement is the most important factor for staging. FFPE sections of cancerous lesions resected surgically from patients with well-characterized clinical history were subjected to laser microdissection (LMD) followed by Liquid Tissue? solubilization and digestion trypsin. Spectral counting was used to measure the amounts of proteins identified by shotgun liquid chromatography (LC)/tandem mass spectrometry (MS/MS). More than 500 proteins were identified from IA and IIIA cases, and non-parametric statistics showed that 81 proteins correlated significantly with stage IA or IIIA. A subset of those proteins were verified by multiple-reaction monitoring mass spectrometric quantitation (MRM assay), described in other paper in this issue. These results demonstrated the technical feasibility of a global proteomic study using clinically well documented FFPE sections, and its possible utility for detailed retrospective disease analyses in order to improve therapeutic strategy.     

Novel In Situ Pretreatment Method for Significantly Enhancing the Signal In MALDI-TOF MS of Formalin-Fixed Paraffin-Embedded Tissue Sections

The application of matrix-assisted laser desorption/ionization (MALDI)-based mass spectrometry (MS) to the proteomic analysis of formalin-fixed paraffin-embedded (FFPE) tissue presents significant technical challenges. In situ enzymatic digestion is frequently used to unlock formalin-fixed tissues for analysis, but the results are often unsatisfactory. Here, we report a new, simplified in situ pretreatment method for preparing tissue sections for MS that involves heating with vapor containing acetonitrile in a small airtight pressurized space. The utility of the novel method is shown using FFPE tissue of human colon carcinoma. The number and intensity of MALDI peaks obtained from analysis of pretreated tissue was significantly higher than control tissue not subjected to pretreatment. A prominent peak (m/z 850) apparently specific to cancerous tissue was identified as a fragment of histone H2A in FFPE tissue pretreated using our method. This highly sensitive treatment may enable MALDI-MS analysis of archived pathological FFPE samples, thus leading to the identification of new biomarkers.     

mTRAQ‐based quantification of potential endometrial carcinoma biomarkers from archived formalin‐fixed paraffin‐embedded tissues

Formalin‐fixed paraffin‐embedded (FFPE) tissues are the primary and preferred medium for archiving patients' samples. Here we demonstrate relative quantifications of protein biomarkers in extracts of laser microdissected epithelial cells from FFPE endometrial carcinoma tissues versus those from normal proliferative endometria by means of targeted proteomic analyses using LC–multiple reaction monitoring (MRM) MS with MRM Tags for Relative and Absolute Quantitation (mTRAQ) labeling. Comparable results of differential expressions for pyruvate kinase isoform M2 (PK‐M2) and polymeric Ig receptor were observed between analyses on laser microdissected epithelial cells from FFPE tissues and corresponding homogenates from frozen tissues of the same individuals that had previously been analyzed and reported. We also identified PK‐M2 in the normal proliferative phase of the endometrium. Other biomarkers in addition to PK‐M2 and polymeric Ig receptor were also observed but not consistently and/or were at levels below the threshold for quantification.     

Expanding the mouse embryonic stem cell proteome: Combining three proteomic approaches

The current study used three different proteomic strategies, which differed by their extent of intact protein separation, to examine the proteome of a pluripotent mouse embryonic stem cell line, R1. Proteins from whole‐cell lysates were subjected either to 2‐D‐LC, or 1‐DE, or were unfractionated prior to enzymatic digestion and subsequent analysis by MS. The results yielded 1895 identified non‐redundant proteins and, for 128 of these, the specific isoform could be determined based on detection of an isoform‐specific peptide. When compared with two previously published proteomic studies that used the same cell line, the current study reveals 612 new proteins.     

Proteomic analysis of liver tissue from HBx‐transgenic mice at early stages of hepatocarcinogenesis

The hepatitis B virus X‐protein (HBx), a multifunctional viral regulator, participates in the viral life cycle and in the development of hepatocellular carcinoma (HCC). We previously reported a high incidence of HCC in transgenic mice expressing HBx. In this study, proteomic analysis was performed to identify proteins that may be involved in hepatocarcinogenesis and/or that could be utilized as early detection biomarkers for HCC. Proteins from the liver tissue of HBx‐transgenic mice at early stages of carcinogenesis (dysplasia and hepatocellular adenoma) were separated by 2‐DE, and quantitative changes were analyzed. A total of 22 spots displaying significant quantitative changes were identified using LC‐MS/MS. In particular, several proteins involved in glucose and fatty acid metabolism, such as mitochondrial 3‐ketoacyl‐CoA thiolase, intestinal fatty acid‐binding protein 2 and cytoplasmic malate dehydrogenase, were differentially expressed, implying that significant metabolic alterations occurred during the early stages of hepatocarcinogenesis. The results of this proteomic analysis provide insights into the mechanism of HBx‐mediated hepatocarcinogenesis. Additionally, this study identifies possible therapeutic targets for HCC diagnosis and novel drug development for treatment of the disease.     

Use of narrow‐range peptide IEF to improve detection of lung adenocarcinoma markers in plasma and pleural effusion

In this study we applied narrow‐range peptide IEF to plasma or pleural effusion prior to LC/MS/MS. Two methods for narrow‐range IEF were run; IPG strips and free‐flow electrophoresis. Data from this study was compared with cell line data to evaluate the method performance in body fluids. To test the methods potential in quantitative biomarker discovery studies, plasma and pleural effusion from patients with lung adenocarcinoma (n=3) were compared with inflammatory pleuritis (n=3) using iTRAQ quantification. Using narrow‐range IEF on the peptide level we were able to identify and quantify 282 proteins in plasma and 300 proteins in pleural effusion. These body fluid proteomes demonstrated high degree of overlap; however, more proteins significantly differently altered levels related to adenenocarcinoma were found in pleural effusion compared with plasma, suggesting enrichment of lung tissue‐related proteins in pleural effusion. Nine proteins were chosen for initial validation with Western blot, and one protein (NPC2) was chosen for further validation using imunohistochemistry. Overall, the quantitative results from IEF/LC/MS/MS showed good correlation with the results from Western blot and imunohistochemistry, showing the potential of this methodology in quantitative biomarker discovery studies.     

Label‐free profiling of skeletal muscle using high‐definition mass spectrometry

We report automated and time‐efficient (2 h per sample) profiling of muscle using ultra‐performance LC coupled directly with high‐definition MS (HDMS^E). Soluble proteins extracted from rat gastrocnemius (n = 10) were digested with trypsin and analyzed in duplicate using a 90 min RPLC gradient. Protein identification and label‐free quantitation were performed from HDMS^E spectra analyzed using Progenesis QI for Proteomics software. In total 1514 proteins were identified. Of these, 811 had at least three unique peptides and were subsequently used to assess the dynamic range and precision of LC‐HDMS^E label‐free profiling. Proteins analyzed by LC‐HDMS^E encompass the entire complement of glycolytic, β‐oxidation, and tricarboxylic acid enzymes. In addition, numerous components of the electron transport chain and protein kinases involved in skeletal muscle regulation were detected. The dynamic range of protein abundances spanned four orders of magnitude. The correlation between technical replicates of the ten biological samples was R² = 0.9961 ± 0.0036 (95% CI = 0.9940 – 0.9992) and the technical CV averaged 7.3 ± 6.7% (95% CI = 6.87 – 7.79%). This represents the most sophisticated label‐free profiling of skeletal muscle to date.