Subnuclear Proteomics in Colorectal Cancer
2010; Elsevier BV; Volume: 9; Issue: 5 Linguagem: Inglês
10.1074/mcp.m900546-mcp200
ISSN1535-9484
AutoresJakob Albrethsen, Jaco C. Knol, Sander R. Piersma, Thang V. Pham, Meike de Wit, Sandra Mongera, Beatriz Carvalho, Henk M.W. Verheul, Remond J.A. Fijneman, Gerrit A. Meijer, Connie R. Jiménez,
Tópico(s)Enzyme Structure and Function
ResumoAbnormalities in nuclear phenotype and chromosome structure are key features of cancer cells. Investigation of the protein determinants of nuclear subfractions in cancer may yield molecular insights into aberrant chromosome function and chromatin organization and in addition may yield biomarkers for early cancer detection. Here we evaluate a proteomics work flow for profiling protein constituents in subnuclear domains in colorectal cancer tissues and apply this work flow to a comparative analysis of the nuclear matrix fraction in colorectal adenoma and carcinoma tissue samples. First, we established the reproducibility of the entire work flow. In a reproducibility analysis of three nuclear matrix fractions independently isolated from the same colon tumor homogenate, 889 of 1,047 proteins (85%) were reproducibly identified at high confidence (minimally two peptides per protein at 99% confidence interval at the protein level) with an average coefficient of variance for the number of normalized spectral counts per protein of 30%. This indicates a good reproducibility of the entire work flow from biochemical isolation to nano-LC-MS/MS analysis. Second, using spectral counting combined with statistics, we identified proteins that are significantly enriched in the nuclear matrix fraction relative to two earlier fractions (the chromatin-binding and intermediate filament fractions) isolated from six colorectal tissue samples. The total data set contained 2,059 non-redundant proteins. Gene ontology mining and protein network analysis of nuclear matrix-enriched proteins revealed enrichment for proteins implicated in "RNA processing" and "mRNA metabolic process." Finally, an explorative comparison of the nuclear matrix proteome in colorectal adenoma and carcinoma tissues revealed many proteins previously implicated in oncogenesis as well as new candidates. A subset of these differentially expressed proteins also exhibited a corresponding change at the mRNA level. Together, the results show that subnuclear proteomics of tumor tissue is feasible and a promising avenue for exploring oncogenesis. Abnormalities in nuclear phenotype and chromosome structure are key features of cancer cells. Investigation of the protein determinants of nuclear subfractions in cancer may yield molecular insights into aberrant chromosome function and chromatin organization and in addition may yield biomarkers for early cancer detection. Here we evaluate a proteomics work flow for profiling protein constituents in subnuclear domains in colorectal cancer tissues and apply this work flow to a comparative analysis of the nuclear matrix fraction in colorectal adenoma and carcinoma tissue samples. First, we established the reproducibility of the entire work flow. In a reproducibility analysis of three nuclear matrix fractions independently isolated from the same colon tumor homogenate, 889 of 1,047 proteins (85%) were reproducibly identified at high confidence (minimally two peptides per protein at 99% confidence interval at the protein level) with an average coefficient of variance for the number of normalized spectral counts per protein of 30%. This indicates a good reproducibility of the entire work flow from biochemical isolation to nano-LC-MS/MS analysis. Second, using spectral counting combined with statistics, we identified proteins that are significantly enriched in the nuclear matrix fraction relative to two earlier fractions (the chromatin-binding and intermediate filament fractions) isolated from six colorectal tissue samples. The total data set contained 2,059 non-redundant proteins. Gene ontology mining and protein network analysis of nuclear matrix-enriched proteins revealed enrichment for proteins implicated in "RNA processing" and "mRNA metabolic process." Finally, an explorative comparison of the nuclear matrix proteome in colorectal adenoma and carcinoma tissues revealed many proteins previously implicated in oncogenesis as well as new candidates. A subset of these differentially expressed proteins also exhibited a corresponding change at the mRNA level. Together, the results show that subnuclear proteomics of tumor tissue is feasible and a promising avenue for exploring oncogenesis. Clinical proteomics aims to comprehensively identify and quantify proteins in human samples to gain insight into cellular pathways in disease and to discover novel biomarkers for screening, early detection, diagnosis, prognosis, and prediction of response to specific treatments. However, a fundamental challenge in clinical proteomics is to overcome the vast dynamic range and complexity of biological proteomes (1Liotta L.A. Kohn E.C. Petricoin E.F. Clinical proteomics: personalized molecular medicine.JAMA. 2001; 286: 2211-2214Crossref PubMed Scopus (195) Google Scholar, 2Jimenez C.R. Piersma S.R. Pham T.V. High-throughput and targeted in-depth mass spectrometry-based approaches for biofluid profiling and biomarker discovery.Biomark. Med. 2007; 4: 541-565Crossref Google Scholar). Some proteomics approaches utilize a combination of multidimensional fractionation and/or depletion of abundant proteins for improved protein coverage. An alternative proteomics strategy is to focus on subproteomes of particular interest, e.g. a subcellular fraction. Subcellular fractionation of tissue and cells in combination with MS/MS has proven to be a powerful approach for the identification of proteins contained in specific organelles, such as the nucleus and mitochondria, as well as other subcellular structures, such as vesicles and the cytoskeleton (3Cox B. Emili A. Tissue subcellular fractionation and protein extraction for use in mass-spectrometry-based proteomics.Nat. Protoc. 2006; 1: 1872-1878Crossref PubMed Scopus (254) Google Scholar, 4Yates 3rd, J.R. Gilchrist A. Howell K.E. Bergeron J.J. Proteomics of organelles and large cellular structures.Nat. Rev. Mol. Cell Biol. 2005; 6: 702-714Crossref PubMed Scopus (340) Google Scholar). Combining MS/MS and subnuclear fractionation provides an even more focused view of the proteomes in selected nuclear subdomains, such as the nuclear envelope (5Schirmer E.C. Florens L. Guan T. Yates 3rd, J.R. Gerace L. Nuclear membrane proteins with potential disease links found by subtractive proteomics.Science. 2003; 301: 1380-1382Crossref PubMed Scopus (513) Google Scholar), the nucleolus (6Andersen J.S. Lyon C.E. Fox A.H. Leung A.K. Lam Y.W. Steen H. Mann M. Lamond A.I. Directed proteomic analysis of the human nucleolus.Curr. Biol. 2002; 12: 1-11Abstract Full Text Full Text PDF PubMed Scopus (805) Google Scholar), and the nuclear matrix (7Albrethsen J. Knol J.C. Jimenez C.R. Unravelling the nuclear matrix proteome.J. Proteomics. 2009; 72: 71-81Crossref PubMed Scopus (51) Google Scholar). Genomic instability is an important feature of colon adenoma to carcinoma progression. About 85% of Colorectal cancer (CRC) 1The abbreviations used are:CRCcolorectal cancerCBchromatin-bindingCVcoefficient of varianceFDRfalse discovery rateGOgene ontologyIFintermediate filamentMARmatrix-associated regionNMnuclear matrixPPIprotein-protein interactionPTMpost-translational modificationMINmicrosatellite instabilityCINchromosome instability2DGEtwo-dimensional gel electrophoresisMIN+MIN-positiveCIN+CIN-positiveMLPAmultiplex ligation-dependent probe amplificationPIPES1,4-piperazinediethanesulfonic acidBis-Tris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolLTQlinear trap quadrupoleIPIInternational Protein IndexSTRINGSearch Tool for the Retrieval of Interacting Genes/ProteinshnRNPheterogeneous nuclear ribonucleoproteinVWFvon Willebrand factorMNDAmyeloid cell nuclear differentiation antigenVCANversican V1HMGB3high mobility group protein B3PDLIMPDZ and LIM domain proteinPPLperiplakinEVPLenvoplakinNUPnuclear pore complex proteinFUBPfar upstream element-binding proteinLTBPlatent transforming growth factor β-binding proteinCHMPcharged multivesicular body proteinPSPC1paraspeckle protein 1PURAPur-αNUPL1nucleoporin-like 1 isoform cTAF15TATA-binding protein-associated factor 2NLAMC1laminin gamma 1VCLvinculinNDFneuregulin 1hnRNPUL1heterogeneous nuclear ribonucleoprotein U-like protein 1YBX1Y-box-binding protein 1SFRSsplicing factor arginine/serine-richU2AF2U2AF 65-kDa subunitNCLnucleolinPIN4peptidyl-prolyl cis-trans isomerase NIMA-interacting 4NUMAnuclear mitotic apparatus proteinNPMnucleophosminGAL3galectin-3CSRP1cysteine- and glycine-rich protein 1RPribosomal proteinNPCnuclear pore complexCTNNcateninGcLCone dimensional gel electrophoresis.1The abbreviations used are:CRCcolorectal cancerCBchromatin-bindingCVcoefficient of varianceFDRfalse discovery rateGOgene ontologyIFintermediate filamentMARmatrix-associated regionNMnuclear matrixPPIprotein-protein interactionPTMpost-translational modificationMINmicrosatellite instabilityCINchromosome instability2DGEtwo-dimensional gel electrophoresisMIN+MIN-positiveCIN+CIN-positiveMLPAmultiplex ligation-dependent probe amplificationPIPES1,4-piperazinediethanesulfonic acidBis-Tris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolLTQlinear trap quadrupoleIPIInternational Protein IndexSTRINGSearch Tool for the Retrieval of Interacting Genes/ProteinshnRNPheterogeneous nuclear ribonucleoproteinVWFvon Willebrand factorMNDAmyeloid cell nuclear differentiation antigenVCANversican V1HMGB3high mobility group protein B3PDLIMPDZ and LIM domain proteinPPLperiplakinEVPLenvoplakinNUPnuclear pore complex proteinFUBPfar upstream element-binding proteinLTBPlatent transforming growth factor β-binding proteinCHMPcharged multivesicular body proteinPSPC1paraspeckle protein 1PURAPur-αNUPL1nucleoporin-like 1 isoform cTAF15TATA-binding protein-associated factor 2NLAMC1laminin gamma 1VCLvinculinNDFneuregulin 1hnRNPUL1heterogeneous nuclear ribonucleoprotein U-like protein 1YBX1Y-box-binding protein 1SFRSsplicing factor arginine/serine-richU2AF2U2AF 65-kDa subunitNCLnucleolinPIN4peptidyl-prolyl cis-trans isomerase NIMA-interacting 4NUMAnuclear mitotic apparatus proteinNPMnucleophosminGAL3galectin-3CSRP1cysteine- and glycine-rich protein 1RPribosomal proteinNPCnuclear pore complexCTNNcateninGcLCone dimensional gel electrophoresis. cases exhibit chromosomal instability (CIN), whereas 15% of CRC cases exhibits microsatellite instability (MIN) (8Lengauer C. Kinzler K.W. Vogelstein B. Genetic instability in colorectal cancers.Nature. 1997; 386: 623-627Crossref PubMed Scopus (1626) Google Scholar). Progression of non-malignant precursor lesions, colorectal adenomas, into CIN+ CRC has been associated with a number of specific chromosomal alterations of which gain of additional copies of chromosome 20q is most prominent (9Hermsen M. Postma C. Baak J. Weiss M. Rapallo A. Sciutto A. Roemen G. Arends J.W. Williams R. Giaretti W. De Goeij A. Meijer G. Colorectal adenoma to carcinoma progression follows multiple pathways of chromosomal instability.Gastroenterology. 2002; 123: 1109-1119Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 10Carvalho B. Postma C. Mongera. S Hopmans E. Diskin S. van de Wiel M.A. van Criekinge W. Thas O. Matthäi A. Cuesta M.A. Terhaar Sive Droste J.S. Craanen M. Schröck E. Ylstra B. Meijer G.A. Multiple putative oncogenes at the chromosome 20q amplicon contribute to colorectal adenoma to carcinoma progression.Gut. 2009; 58: 79-89Crossref PubMed Scopus (190) Google Scholar). Tumor progression is accompanied by important phenotypic changes in neoplastic cells, in particular alterations of nuclear structure such as nuclear size and shape, numbers and sizes of nucleoli, and chromatin texture as observed by the pathologist under the microscope (9Hermsen M. Postma C. Baak J. Weiss M. Rapallo A. Sciutto A. Roemen G. Arends J.W. Williams R. Giaretti W. De Goeij A. Meijer G. Colorectal adenoma to carcinoma progression follows multiple pathways of chromosomal instability.Gastroenterology. 2002; 123: 1109-1119Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 11Pienta K.J. Partin A.W. Coffey D.S. Cancer as a disease of DNA organization and dynamic cell structure.Cancer Res. 1989; 49: 2525-2532PubMed Google Scholar, 12Zink D. Fischer A.H. Nickerson J.A. Nuclear structure in cancer cells.Nat. Rev. Cancer. 2004; 4: 677-687Crossref PubMed Scopus (716) Google Scholar). Yet, still little is known about the protein constituents underlying these phenotypic characteristics (12Zink D. Fischer A.H. Nickerson J.A. Nuclear structure in cancer cells.Nat. Rev. Cancer. 2004; 4: 677-687Crossref PubMed Scopus (716) Google Scholar). As such, proteins supporting nuclear structure and organization represent a relevant target for proteomics analysis of cancer tissue and precursor lesions. Here we evaluate a proteomics work flow for profiling of subnuclear fractions from early stage CRC tumors and apply this work flow to a comparative analysis of the nuclear matrix fraction of colorectal adenoma and carcinoma tissues. colorectal cancer chromatin-binding coefficient of variance false discovery rate gene ontology intermediate filament matrix-associated region nuclear matrix protein-protein interaction post-translational modification microsatellite instability chromosome instability two-dimensional gel electrophoresis MIN-positive CIN-positive multiplex ligation-dependent probe amplification 1,4-piperazinediethanesulfonic acid 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol linear trap quadrupole International Protein Index Search Tool for the Retrieval of Interacting Genes/Proteins heterogeneous nuclear ribonucleoprotein von Willebrand factor myeloid cell nuclear differentiation antigen versican V1 high mobility group protein B3 PDZ and LIM domain protein periplakin envoplakin nuclear pore complex protein far upstream element-binding protein latent transforming growth factor β-binding protein charged multivesicular body protein paraspeckle protein 1 Pur-α nucleoporin-like 1 isoform c TATA-binding protein-associated factor 2N laminin gamma 1 vinculin neuregulin 1 heterogeneous nuclear ribonucleoprotein U-like protein 1 Y-box-binding protein 1 splicing factor arginine/serine-rich U2AF 65-kDa subunit nucleolin peptidyl-prolyl cis-trans isomerase NIMA-interacting 4 nuclear mitotic apparatus protein nucleophosmin galectin-3 cysteine- and glycine-rich protein 1 ribosomal protein nuclear pore complex catenin one dimensional gel electrophoresis. colorectal cancer chromatin-binding coefficient of variance false discovery rate gene ontology intermediate filament matrix-associated region nuclear matrix protein-protein interaction post-translational modification microsatellite instability chromosome instability two-dimensional gel electrophoresis MIN-positive CIN-positive multiplex ligation-dependent probe amplification 1,4-piperazinediethanesulfonic acid 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol linear trap quadrupole International Protein Index Search Tool for the Retrieval of Interacting Genes/Proteins heterogeneous nuclear ribonucleoprotein von Willebrand factor myeloid cell nuclear differentiation antigen versican V1 high mobility group protein B3 PDZ and LIM domain protein periplakin envoplakin nuclear pore complex protein far upstream element-binding protein latent transforming growth factor β-binding protein charged multivesicular body protein paraspeckle protein 1 Pur-α nucleoporin-like 1 isoform c TATA-binding protein-associated factor 2N laminin gamma 1 vinculin neuregulin 1 heterogeneous nuclear ribonucleoprotein U-like protein 1 Y-box-binding protein 1 splicing factor arginine/serine-rich U2AF 65-kDa subunit nucleolin peptidyl-prolyl cis-trans isomerase NIMA-interacting 4 nuclear mitotic apparatus protein nucleophosmin galectin-3 cysteine- and glycine-rich protein 1 ribosomal protein nuclear pore complex catenin one dimensional gel electrophoresis. A focused proteomics work flow for analysis of patient tumor tissue should effectively separate subproteomes of interest from interfering and highly abundant proteins and other tissue components. In addition, the method must be practical, reproducible, and compatible with relatively small amounts of tissue. Here we investigate a biochemical protocol that originally was developed by Fey and co-workers (13Fey E.G. Wan K.M. Penman S. Epithelial cytoskeletal framework and nuclear matrix-intermediate filament scaffold: three-dimensional organization and protein composition.J. Cell Biol. 1984; 98: 1973-1984Crossref PubMed Scopus (425) Google Scholar, 14Fey E.G. Penman S. Nuclear matrix proteins reflect cell type of origin in cultured human cells.Proc. Natl. Acad. Sci. U.S.A. 1988; 85: 121-125Crossref PubMed Scopus (277) Google Scholar) in the 1980s. In brief, tissue samples are homogenized in a solution with stabilizers of RNA-protein complexes, and soluble, cytoskeletal, and DNA-associated proteins are extracted. The remaining detergent-, salt-, and DNase-resistant pellet is cleared of filaments and is referred to as the "nuclear matrix" (NM) fraction. The NM has been postulated to be a dynamic RNA/protein network that spans the nucleoplasm and to provide support for higher order chromatin packaging and overall shape of the nucleus (15Nickerson J.A. Krockmalnic G. Wan K.M. Penman S. The nuclear matrix revealed by eluting chromatin from a cross-linked nucleus.Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 4446-4450Crossref PubMed Scopus (126) Google Scholar). Several lines of evidence support roles for the NM in the processing of genetic information and for establishing functional subdomains and macromolecule assemblies in the nucleus (15Nickerson J.A. Krockmalnic G. Wan K.M. Penman S. The nuclear matrix revealed by eluting chromatin from a cross-linked nucleus.Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 4446-4450Crossref PubMed Scopus (126) Google Scholar). Oncogenes or suppressor genes can function differentially in different cell types, and much of this cellular context for transformation may be determined, among others, by the NM (16Coffey D.S. Nuclear matrix proteins as proteomic markers of preneoplastic and cancer lesions.Clin. Cancer Res. 2002; 8: 3031-3033PubMed Google Scholar). Interestingly, early studies using two-dimensional gel-based proteomics have indicated that the NM proteome varies significantly between different tissues and cells and in cancer versus healthy tissue (for reviews, see Refs. 7Albrethsen J. Knol J.C. Jimenez C.R. Unravelling the nuclear matrix proteome.J. Proteomics. 2009; 72: 71-81Crossref PubMed Scopus (51) Google Scholar and 17Leman E.S. Getzenberg R.H. Nuclear structure as a source of cancer specific biomarkers.J. Cell. Biochem. 2008; 104: 1988-1993Crossref PubMed Scopus (27) Google Scholar). However, only a few cancer-related NM proteins have been identified so far. A comprehensive LC-MS/MS-based proteomics analysis of the NM proteome in adenoma-carcinoma progression may shed light on the potential role of the NM in (tissue-specific) cancer pathogenesis; in particular, the NM proteins have been suggested to provide a promising source of protein biomarkers for cancer (17Leman E.S. Getzenberg R.H. Nuclear structure as a source of cancer specific biomarkers.J. Cell. Biochem. 2008; 104: 1988-1993Crossref PubMed Scopus (27) Google Scholar, 18Sjakste N. Sjakste T. Vikmanis U. Role of the nuclear matrix proteins in malignant transformation and cancer diagnosis.Exp. Oncol. 2004; 26: 170-178PubMed Google Scholar). The NMP22/Bladdercheck (Matritech) test measures urine levels of the NM-associated protein nuclear mitotic apparatus protein (NUMA) and has been United States Food and Drug Administration-approved for follow-up measurements in bladder cancer patients (17Leman E.S. Getzenberg R.H. Nuclear structure as a source of cancer specific biomarkers.J. Cell. Biochem. 2008; 104: 1988-1993Crossref PubMed Scopus (27) Google Scholar). In addition to the NM fraction, two hitherto uncharacterized fractions are also extracted with this protocol: the "chromatin-binding" (CB) and the "intermediate filament" (IF) fractions. Using differential analysis of the three fractions, we show that the NM fraction is enriched for proteins derived from distinct subnuclear domains. Importantly, this subcellular fractionation approach was reproducible, and a differential comparison of the nuclear matrix proteome in colorectal adenoma and carcinoma tissues uncovered many proteins previously implicated in cancer biology as well as novel candidates. Therefore, the proteomics work flow presented here provides relevant targets for comprehensive profiling of proteins involved in subnuclear structure and organization in early stage CRC tumors and is a promising avenue to explore oncogenesis. All basic chemicals were obtained from Sigma. HPLC solvents, LC-MS grade water, acetonitrile, and formic acid were obtained from Biosolve (Valkenswaard, The Netherlands). Porcine sequence grade modified trypsin was obtained from Promega (Leiden, The Netherlands). Complete protease inhibitor mixture tablets (mini, EDTA-free) were purchased from Roche Diagnostics. Protein assay reagent was obtained from Bio-Rad. Precast gradient gels and gel buffers were acquired from Invitrogen. CRC tissue was collected at the VU University Medical Center, Amsterdam, The Netherlands, in compliance with institutional regulations for use of leftover material. The tissue samples (50–200 mg) were obtained from surgical resection specimens at the department of pathology, snap frozen in liquid nitrogen, and stored at −80 °C. All samples were inspected by a pathologist to ascertain a tumor content of at least 70% cancer cells using hematoxylin- and eosin-stained sections. Biological subtypes of carcinomas (MIN+ versus CIN+) were determined to include both carcinoma types in the analysis. In brief, copy number changes were determined by MLPA as described by Schouten et al. (19Schouten J.P. McElgunn C.J. Waaijer R. Zwijnenburg D. Diepvens F. Pals G. Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification.Nucleic Acids Res. 2002; 30: e57Crossref PubMed Scopus (2067) Google Scholar). To this end, we used previously designed probes for chromosomes arms 8q, 13q, and 20q (20Buffart T.E. van Grieken N.C. Tijssen M. Coffa J. Ylstra B. Grabsch H.I. van de Velde C.J. Carvalho B. Meijer G.A. High resolution analysis of DNA copy-number aberrations of chromosomes 8, 13, and 20 in gastric cancers.Virchows Arch. 2009; 455: 213-223Crossref PubMed Scopus (51) Google Scholar). MLPA data analysis was performed using MLPAnalyzer version 7.0 (21Coffa J. van de Wiel M.A. Diosdado B. Carvalho B. Schouten J. Meijer G.A. MLPAnalyzer: data analysis tool for reliable automated normalization of MLPA fragment data.Cell. Oncol. 2008; 30: 323-335PubMed Google Scholar). MIN analysis was performed using the MIN Analysis System (MSI Multiplex System Version 1.1, Promega) as described (22Derks S. Postma C. Carvalho B. van den Bosch S.M. Moerkerk P.T. Herman J.G. Weijenberg M.P. de Bruïne A.P. Meijer G.A. van Engeland M. Integrated analysis of chromosomal, microsatellite and epigenetic instability in colorectal cancer identifies specific associations between promoter methylation of pivotal tumour suppressor and DNA repair genes and specific chromosomal alterations.Carcinogenesis. 2008; 29: 434-439Crossref PubMed Scopus (56) Google Scholar). Clinical details are described in supplemental Table 1. The tissue was prepared essentially by the protocol originally developed by Fey and co-workers (13Fey E.G. Wan K.M. Penman S. Epithelial cytoskeletal framework and nuclear matrix-intermediate filament scaffold: three-dimensional organization and protein composition.J. Cell Biol. 1984; 98: 1973-1984Crossref PubMed Scopus (425) Google Scholar, 14Fey E.G. Penman S. Nuclear matrix proteins reflect cell type of origin in cultured human cells.Proc. Natl. Acad. Sci. U.S.A. 1988; 85: 121-125Crossref PubMed Scopus (277) Google Scholar). The homogenization and fractionation were performed in a cold room (4 °C) and on ice. First, the frozen tissue was placed in a Petri dish with ∼1 ml of liquid nitrogen and cut into small pieces (∼1 mm2) with a precooled razor blade. The frozen tissue pieces were immediately placed in a precooled Potter-Elvehjem tube containing 600 µl of precooled homogenization buffer/100 mg of tissue (0.5% Triton X-100, 300 mm sucrose, 100 mm NaCl, 10 mm PIPES, pH 6.8, 3 mm MgCl2, 2 mm vanadyl ribonucleoside, 1 mm EGTA, 1 mm PMSF, Complete protease inhibitor mixture (Roche Diagnostics)). The tissue was homogenized for 2 min (50 strokes) on ice using a precooled Potter-Elvehjem homogenizer and electrical drill. Next, the homogenate was filtered through three layers of 350-µm nylon mesh. The homogenate was left to mix on a turning wheel for 5 min (4 °C) and centrifuged for 5 min at 750 × g (4 °C), and the supernatant was collected (fraction 1, the "cytosolic" fraction containing lipids and soluble proteins). The pellet was resuspended in 600 µl of "extraction buffer"/100 mg of starting material (same as homogenization buffer but with 250 mm ammonium sulfate instead of 100 mm NaCl). The sample was mixed on a turning wheel for 5 min (4 °C) and centrifuged for 5 min at 750 × g (4 °C), and the supernatant was collected (fraction 2, the "cytoskeletal" fraction containing salt-extracted tissue elements and proteins). Third, the remaining pellet was resuspended in 400 µl of "digestion buffer"/100 mg of starting tissue (same as homogenization buffer but with 100 µg/ml RNase-free DNase I, and a reduced concentration of 50 mm NaCl). The sample was left at room temperature for 30 min for digestion of chromosomal DNA. The sample was brought back to high-salt conditions by addition of ammonium sulfate solution, left at room temperature for 5 min, centrifuged for 5 min at 1000 g (4 °C), and the supernatant was collected (fraction 3, the chromatin-binding fraction). The remaining detergent-, high salt-, and DNase-resistant pellet was resuspended in 400 µl of "disassembly buffer"/100 mg of starting material (8 m urea, 20 mm PIPES, pH 6.8, 1 mm EGTA, 1 mm DTT, 1 mm PMSF). The suspended proteins were treated by overnight dialysis against >1,000 volumes of "reassembly buffer" (25 mm imidazole, pH 7.1, 150 mm KCl, 5 mm MgCl2, 0.125 mm EGTA, 1.4 mm DTT, 0.26 mm PMSF). The repolymerized filaments were pelleted by centrifugation for 90 min at 217,000 × g at 20 °C. The visible pellet was dissolved in Tris buffer (10 mm Tris-HCl, pH 7.0, 100 mm KCl), shaken vigorously, and incubated overnight at 4 °C after which the majority of protein was dissolved (fraction 4, the intermediate filament fraction). The 217,000 × g supernatant was collected as the final fraction (fraction 5, the nuclear matrix fraction). For the analysis of unfractionated CRC tissue, three CRC tissue samples (∼75 mg each) were homogenized in a ratio of 100 mg/1 ml of buffer (7 m urea, 2 m thiourea, 4% CHAPS, and 10 µl/ml protease inhibitor mixture). Protein concentrations of the samples were determined with the Bradford assay (Bio-Rad). For proteomics analysis, 60 µg of CB, IF, and NM fractions and unfractionated CRC lysates were loaded on SDS-polyacrylamide gels. SDS-PAGE was performed with precast 4–12% gradient gels containing Bis-Tris buffer (NuPAGE MES system, Invitrogen). After electrophoresis, the gels were fixed in 50% ethanol containing 3% phosphoric acid and stained with Coomassie Blue R-250. After staining, the gels were washed in Milli-Q water and stored at 4 °C until processing for in-gel digestion. Coomassie-stained entire gel lanes were cut in 10 bands, and each band was processed for in-gel digestion according to the method of Shevchenko et al. (23Shevchenko A. Wilm M. Vorm O. Mann M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels.Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7735) Google Scholar). Briefly, bands were washed and dehydrated three times in 50 mm ammonium bicarbonate, pH 7.9 and in 50 mm ammonium bicarbonate, pH 7.9 + 50% ACN, respectively. Subsequently, cysteine bonds were reduced with 10 mm DTT for 1 h at 56 °C and alkylated with 50 mm iodoacetamide for 45 min at room temperature in the dark. After two subsequent wash/dehydration cycles, the bands were dried for 10 min in a vacuum centrifuge and incubated overnight with 0.06 µg/µl trypsin at 25 °C. Peptides were extracted once in 1% formic acid and subsequently two times in 50% ACN in 5% formic acid. The volume was reduced to 50 µl in a vacuum centrifuge prior to nano-LC-MS/MS analysis. Peptides were separated by an Ultimate 3000 nano-LC system (Dionex LC Packings, Amsterdam, The Netherlands) equipped with a 20 cm × 75-µm inner diameter fused silica column custom-packed with 3-µm 120-Å ReproSil Pur C18 aqua (Dr. Maisch GmbH, Ammerbuch-Entringen, Germany). After injection, peptides were trapped at 30 µl/min on a 5 mm × 300-µm inner diameter PepMap C18 cartridge (Dionex LC Packings) at 2% buffer B (buffer A, 0.05% formic acid in water; buffer B, 80% ACN + 0.05% formic acid in Milli-Q) and separated at 300 nl/min in a 10–40% buffer B gradient in 60 min. Nano-LC-MS/MS data were acquired for the three subnuclear compartments (CB, IF, and NMP) in three batches with six colorectal tissues within 3 months and stable instrument performance. Per subnuclear fraction, the injection scheme of the 10 gel bands per sample was as follows: "adenoma1-band1," "adenoma2-band1," "MIN1-band1," "MIN2-band1," "CIN1-band1," and "CIN2-band1" followed by the second gel band for all samples, the third, etc. until band 10. The injection scheme spreads instrumental drift over the biological samples and minimizes bias. Eluting peptides were ionized at 1.7 kV in a Nanomate Triversa Chip-based nanospray source using a Triversa LC coupler (Advion, Ithaca, NY). Intact peptide mass spectra and fragmentation spectra were acquired on an LTQ-FT hybrid mass spectrometer (Thermo Fi
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