An Informatics-assisted Label-free Approach for Personalized Tissue Membrane Proteomics: Case Study on Colorectal Cancer
2011; Elsevier BV; Volume: 10; Issue: 4 Linguagem: Inglês
10.1074/mcp.m110.003087
ISSN1535-9484
AutoresChia‐Li Han, Jinn-Shiun Chen, Err–Cheng Chan, Chien-Peng Wu, Kun‐Hsing Yu, Kuei-Tien Chen, Chih‐Chiang Tsou, Chia-Feng Tsai, Chih-Wei Chien, Yung-Bin Kuo, Pei-Yi Lin, Jau‐Song Yu, Chuen Hsueh, Min-Chi Chen, Chung-Chuan Chan, Yu‐Sun Chang, Yu‐Ju Chen,
Tópico(s)Cell Adhesion Molecules Research
ResumoWe developed a multiplexed label-free quantification strategy, which integrates an efficient gel-assisted digestion protocol, high-performance liquid chromatography tandem MS analysis, and a bioinformatics alignment method to determine personalized proteomic profiles for membrane proteins in human tissues. This strategy provided accurate (6% error) and reproducible (34% relative S.D.) quantification of three independently purified membrane fractions from the same human colorectal cancer (CRC) tissue. Using CRC as a model, we constructed the personalized membrane protein atlas of paired tumor and adjacent normal tissues from 28 patients with different stages of CRC. Without fractionation, this strategy confidently quantified 856 proteins (≥2 unique peptides) across different patients, including the first and robust detection (Mascot score: 22,074) of the well-documented CRC marker, carcinoembryonic antigen 5 by a discovery-type proteomics approach. Further validation of a panel of proteins, annexin A4, neutrophils defensin A1, and claudin 3, confirmed differential expression levels and high occurrences (48–70%) in 60 CRC patients. The most significant discovery is the overexpression of stomatin-like 2 (STOML2) for early diagnostic and prognostic potential. Increased expression of STOML2 was associated with decreased CRC-related survival; the mean survival period was 34.77 ± 2.03 months in patients with high STOML2 expression, whereas 53.67 ± 3.46 months was obtained for patients with low STOML2 expression. Further analysis by ELISA verified that plasma concentrations of STOML2 in early-stage CRC patients were elevated as compared with those of healthy individuals (p < 0.001), suggesting that STOML2 may be a noninvasive serological biomarker for early CRC diagnosis. The overall sensitivity of STOML2 for CRC detection was 71%, which increased to 87% when combined with CEA measurements. This study demonstrated a sensitive, label-free strategy for differential analysis of tissue membrane proteome, which may provide a roadmap for the subsequent identification of molecular target candidates of multiple cancer types. We developed a multiplexed label-free quantification strategy, which integrates an efficient gel-assisted digestion protocol, high-performance liquid chromatography tandem MS analysis, and a bioinformatics alignment method to determine personalized proteomic profiles for membrane proteins in human tissues. This strategy provided accurate (6% error) and reproducible (34% relative S.D.) quantification of three independently purified membrane fractions from the same human colorectal cancer (CRC) tissue. Using CRC as a model, we constructed the personalized membrane protein atlas of paired tumor and adjacent normal tissues from 28 patients with different stages of CRC. Without fractionation, this strategy confidently quantified 856 proteins (≥2 unique peptides) across different patients, including the first and robust detection (Mascot score: 22,074) of the well-documented CRC marker, carcinoembryonic antigen 5 by a discovery-type proteomics approach. Further validation of a panel of proteins, annexin A4, neutrophils defensin A1, and claudin 3, confirmed differential expression levels and high occurrences (48–70%) in 60 CRC patients. The most significant discovery is the overexpression of stomatin-like 2 (STOML2) for early diagnostic and prognostic potential. Increased expression of STOML2 was associated with decreased CRC-related survival; the mean survival period was 34.77 ± 2.03 months in patients with high STOML2 expression, whereas 53.67 ± 3.46 months was obtained for patients with low STOML2 expression. Further analysis by ELISA verified that plasma concentrations of STOML2 in early-stage CRC patients were elevated as compared with those of healthy individuals (p < 0.001), suggesting that STOML2 may be a noninvasive serological biomarker for early CRC diagnosis. The overall sensitivity of STOML2 for CRC detection was 71%, which increased to 87% when combined with CEA measurements. This study demonstrated a sensitive, label-free strategy for differential analysis of tissue membrane proteome, which may provide a roadmap for the subsequent identification of molecular target candidates of multiple cancer types. Colorectal cancer (CRC) 1The abbreviations used are:CRCcolorectal cancerS.D.standard deviationANXA4annexin A4DEFA1neutrophil defensin A1CLDN3claudin 3STOML2stomatin-like 2CEAcarcinoembryonic antigenIHCimmunohistochemical stainingXICextracted ion chromatographyROCreceiver operating characteristicAUCthe area under the ROC curve. is one of the most prevalent cancers and the fourth leading cause of cancer mortality worldwide, with an estimated 1,000,000 new cases and ∼500,000 related deaths every year (1Weitz J. Koch M. Debus J. Höhler T. Galle P.R. Büchler M.W. Colorectal cancer.Lancet. 2005; 365: 153-165Abstract Full Text Full Text PDF PubMed Scopus (1033) Google Scholar, 2Jemal A. Siegel R. Ward E. Murray T. Xu J. Thun M.J. Cancer statistics, 2007.CA Cancer J. Clin. 2007; 57: 43-66Crossref PubMed Scopus (7502) Google Scholar). Detection of CRC and subsequent intervention at an earlier stage has the potential to reduce both incidence and mortality of the disease (3Kronborg O. Fenger C. 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[Colorectal cancer 2D-proteomics: identification of altered protein expression].Mol. Biol. 2009; 43: 348-356Google Scholar). By analyzing paired tumor and adjacent normal tissues from patients, these gel-based methods have led to discovery of a variety of proteins involving in signal transduction, cellular reorganization, and tissue hypoxia as potential biomarkers for CRC. Alfonso et al. (25Alfonso P. Cañamero M. Fernández-Carbonié F. Núñez A. Casal J.I. Proteome analysis of membrane fractions in colorectal carcinomas by using 2D-DIGE saturation labeling.J. Proteome Res. 2008; 7: 4247-4255Crossref PubMed Scopus (78) Google Scholar) analyzed the membrane fractions of six paired CRC mucosal tissues using two-dimensional differential gel electrophoresis analysis, identifying annexin A2, annexin A4 (ANXA4) and VDAC as potential markers for CRC diagnosis and, presumably, therapy. In a recent study, Ma et al. (20Ma Y. Peng J. Liu W. Zhang P. Huang L. Gao B. Shen T. Zhou Y. Chen H. Chu Z. 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A multiplexed quantitative strategy for membrane proteomics: opportunities for mining therapeutic targets for autosomal dominant polycystic kidney disease.Mol. Cell Proteomics. 2008; 7: 1983-1997Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar), high-performance liquid chromatography tandem MS (LC-MS/MS) analysis, and a bioinformatics alignment method (32Tsou C.C. Tsai C.F. Tsui Y.H. Sudhir P.R. Wang Y.T. Chen Y.J. Chen J.Y. Sung T.Y. Hsu W.L. IDEAL-Q, an automated tool for label-free quantitation analysis using an efficient peptide alignment approach and spectral data validation.Mol. Cell Proteomics. 2010; 9: 131-144Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar) in attempt to comprehensively map and accurately quantify the membrane proteome. Using CRC as a model to discover potential biomarkers for diagnosis, we applied this strategy to the analysis of differentially expressed membrane proteins in tumor and adjacent normal tissues from each CRC patient. Twenty-eight paired tumor and adjacent normal tissues from patients in Dukes' A (n = 4), Dukes' B (n = 7), Dukes' C (n = 11) and Dukes' D (n = 6) stages were analyzed. We addressed two issues. (1) From the technical prospective, can this approach provide good sensitivity for robust identification of the current CRC protein biomarker, CEA (CEACAM5)? (2) More importantly, can the technological advancement lead to discovery of additional proteins that are involved in colon tumorigenesis and that can serve as new diagnostic and prognostic protein biomarkers? Contrary to most previous studies using pooled tissue samples, the individual paired-tissue comparisons used here provide information concerning biological and genetic variations between different individuals. The high-throughput strategy is also advantageous in that it provides an analysis of individual proteomic patterns among patients to evaluate the heterogeneity of tissues profiles. In addition to the confident identification of CEA in the 28 patients, this study identified a panel of membrane proteins with high levels of elevated expression levels in CRC patients. To further evaluate the clinical relevance of these candidates, their expression levels in tissue or serum were examined by Western blot, immunohistochemical staining, ELISA, and clinicopathologic analysis from a large cohort of patients with known clinical outcomes. The results demonstrated the power of tissue membrane proteomics for the discovery of valuable biomarker candidates for early diagnosis and prognosis of CRC. Protease inhibitor was obtained from Merck (Darmstadt, Germany). Monomeric acrylamide/bisacrylamide solution (40%, 29:1) was purchased from Bio-Rad (Hercules, CA). Trypsin (modified, sequencing grade) was obtained from Promega (Madison, WI). The BCA and Bradford protein assay reagent kits were obtained from Pierce (Rockford, IL). SDS was purchased from GE Healthcare (Central Plaza, Singapore). Ammonium persulfate (APS) and N,N,N′,N′-tetramethylethylenediamine (TEMED) were purchased from Amersham Biosciences (Piscataway, NJ). Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), triethylammonium bicarbonate (TEABC), methyl methanethiosulfonate (MMTS), trifluoroacetic acid (TFA), sodium carbonate (Na2CO3), sucrose, Tris-HCl, NaCl, MgCl2, and HPLC-grade acetonitrile (ACN) were purchased from Sigma-Aldrich (St. Louis, MO). Formic acid (FA) was purchased from Riedel de Haen (Seelze, Germany). Water was obtained from a Milli-Q Ultrapure Water Purification System (Millipore, Billerica, MA). Clinical tissue samples were obtained from Chang Gung Memorial Hospital at Lin-Kou, Taiwan in accordance with approved human subject guidelines authorized by Medical Ethics and Human Clinical Trial Committee at Chang Gung Memorial Hospital. Following surgery, the tumor and adjacent normal tissues were collected in separate tubes, kept on dry ice for 30 min during transportation, and stored at −80 °C before further processing. Adjacent normal tissue was obtained from the distal edge of the resection ≥10 cm from the tumor. In the discovery phase, a total of 28 pairs of cancerous and adjacent normal tissue were collected and analyzed from individual patients with Dukes' A (n = 4), Dukes' B (n = 7), Dukes' C (n = 11) or Dukes' D (n = 6) stages CRC patients (supplemental Table 1). In the validation phase, 205 colorectal carcinomas patients and 140 blood samples without hemolysis or lipemia, including 70 samples from CRC patient and 70 age-matched individuals without CRC, were collected from the Department of Colorectal Cancer, Chang Gung Memorial Hospital (33Wu C.C. Hsu C.W. Chen C.D. Yu C.J. Chang K.P. Tai D.I. Liu H.P. Su W.H. Chang Y.S. Yu J.S. Candidate serological biomarkers for cancer identified from the secretomes of 23 cancer cell lines and the human protein atlas.Mol. Cell Proteomics. 2010; 9: 1100-1117Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). All CRC patients had histologically verified adenocarcinoma of the colon or rectum that was confirmed by pathologists. Patient characteristics were obtained from pathology records. Subjects with a history of other malignant diseases or infectious disease, or who had undergone surgery 6 months prior to the start of this research were excluded for this retrospective study. Fresh plasma samples were obtained before surgery and were stored at −80 °C until use. Frozen tissues were thawed rapidly at 37 °C, cut into small pieces, and washed by 0.9% NaCl to remove blood. The precleaned tissues were homogenized in STM solution (5 ml/g tissue, 0.25 m sucrose, 10 mm Tris-HCl, and 1 mm MgCl2) with protease inhibitor mixture (100:1, sample/protease inhibitor, v/v, Calbiochem) using homogenizer mechanism (Polytron System PT 1200 E, Luzernerstrasse, Switzerland). Nuclei and tissue debris were removed by centrifugation (260 × g) for 5 min at 4 °C. The supernatant was first centrifuged at 1500 × g for 10 min at 4 °C to pellet the crude membrane proteins. The pellet was mixed with two-thirds of the original homogenate volume (0.25 m STM solution with protease inhibitor mixture) and then centrifuged at 16,000 × g for 1 h at 4 °C to purify the membrane pellet. The pellet was washed in 1 ml of 0.1 m Na2CO3 for overnight at 4 °C and re-collected by centrifugation at 16,000 × g for 1 h at 4 °C. The purified membrane pellet was dissolved in 50 μl of 90% (v/v) FA prior to the Bradford assay to determine the membrane protein concentration and then was vacuum dried and stored at −80 °C for further processing. The membrane protein pellet was subjected to our previously reported gel-assisted digestion (31Han C.L. Chien C.W. Chen W.C. Chen Y.R. Wu C.P. Li H. Chen Y.J. A multiplexed quantitative strategy for membrane proteomics: opportunities for mining therapeutic targets for autosomal dominant polycystic kidney disease.Mol. Cell Proteomics. 2008; 7: 1983-1997Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). In brief, membrane protein pellet was resuspended in 6 m urea, 5 mm EDTA, and 2% (v/v) SDS in 0.1 m TEABC and sonicated by a Bioruptor (Diagenode, Belgium) at 4 °C for 10 min. Bovine serum Albumin (BSA) was added as internal standard (1000:1, protein/BSA, w/w). Proteins were reduced by 5 mm TCEP and alkylated by 2 mm MMTS at room temperature for 30 min. Acrylamide/bisacrylamide (40%, 29:1, v/v), 10% (w/v) APS, and 100% TEMED were then applied to the sample to polymerize as a gel directly in the microcentrifuge without electrophoresis. The gel was cut into small pieces, washed several times (0.1 m TEABC in 50% (v/v) ACN) and subjected to tryptic digestion (10:1, protein/trypsin, w/w) in 25 mm TEABC overnight at 37 °C. Peptides were extracted from the gel using sequential extraction with 25 mm TEABC, 0.1% (v/v) TFA in water, 0.1% (v/v) TFA in ACN, and 100% ACN. The extracted peptides were concentrated in a SpeedVac (Thermo Savant SC210A, Holbrook, NY), desalted by using C18 ZipTip (Millipore; Cambridge, Ontario, Canada), and subjected to LC-MS/MS analysis. Peptide samples were reconstituted in 0.1% (v/v) FA in H2O and analyzed by Waters Q-TOFTM Premier (Waters Corp., Milford, MA). Samples were injected into a 20-mm × 180-μm trap column, separated by 200-mm × 75 mm Waters1 ACQUITY 1.7 mm BEH C18 column using a nanoACQUITY Ultra Performance LCTM system (Waters Corp., Milford, MA), and eluted with a linear gradient of 0–80% of 0.1% (v/v) FA in ACN for 120 min at 300 nl/min. MS was operated in electrospray ionization positive V mode with a resolving power of 10,000. A NanoLockSprayTM source (Waters Corp., Milford, MA) was used for accurate mass measurements, and the lock mass channel was sampled every 30 s. The mass spectrometer was calibrated with a synthetic human [Glu1]-Fibrinopeptide B solution (1 pmol/μl; Sigma Aldrich) delivered through the NanoLockSpray source. Data acquisition was operated in the data-directed analysis mode to automatically switch between a full MS scan (m/z 400–1600, 0.6 s) and three MS/MS scans (m/z 100–1990, 1.2 s for each scan) sequentially on the three most intense ions present in the full MS scan. The peak list resulting from MS/MS spectra was exported to mgf format by Mascot Distiller v2.1.1.0. The datasets were batch-searched and combined-searched by Mascot v2.2 (Matrix science, London, United Kingdom) against International Protein Index (IPI) human database (34Kersey P.J. Duarte J. Williams A. Karavidopoulou Y. Birney E. Apweiler R. The International Protein Index: an integrated database for proteomics experiments.Proteomics. 2004; 4: 1985-1988Crossref PubMed Scopus (640) Google Scholar) (v3.29, 68161 sequences) from the European Bioinformatics Institute using the following constraints: only tryptic peptides with up to two missed cleavage sites were allowed; 0.3-Da mass tolerances for MS and 0.1-Da mass tolerances for MS/MS fragment ions. Methylthio (Cys) and oxidation (Met) was specified as variable modifications. Only unique peptides with scores ≥35 (p < 0.05) were confidently assigned. In each MS/MS spectrum, a total of at least four b- and y-ions were observed. To evaluate the false discovery rate in protein identification, we performed a decoy database search against a randomized decoy database created by Mascot using identical search parameters and validation criteria. The search results in Mascot were exported in eXtensive Markup Language data format (XML). The MS/MS spectra and assignment for single peptide identification are included in supplemental Fig. 1. For label-free quantification, data analysis was performed by our recently developed IDEAL-Q software (32Tsou C.C. Tsai C.F. Tsui Y.H. Sudhir P.R. Wang Y.T. Chen Y.J. Chen J.Y. Sung T.Y. Hsu W.L. IDEAL-Q, an automated tool for label-free quantitation analysis using an efficient peptide alignment approach and spectral data validation.Mol. Cell Proteomics. 2010; 9: 131-144Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). The raw data files, from Waters Q-TOF Premier were converted into files of mzXML format by masswolf v4.0. IDEAL-Q performs quantitation analysis using spectral data in mzXML format and Mascot search result in XML format. The abundance of a peptide was determined by the extracted ion chromatography (XIC) and further normalized by XIC of the internal standard peptide. The protein ratio was determined by a weighted average of the peptide ratios, where the wei
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