[en] Formalin-fixed paraffin-embedded (FFPE) specimens of patients are useful sources of materials for clinical research and have recently gained interest for use in the discovery of clinical proteomic biomarkers. However, the critical step in this field is the ability to obtain an efficient and repeatable extraction using the limited quantities of material available for research in hospital biobanks. This work describes the evaluation of the peptide/protein extraction using FFPE sections treated by the following two methods before shotgun proteomic analysis: a commercial solution (FFPE-FASP) (filter aided sample preparation) and an antigen retrieval-derived protocol (On Slice AR). Their efficiencies and repeatabilities are compared using data-independent differential quantitative label-free analysis. FFPE-FASP was shown to be globally better both qualitatively and quantitatively than On Slice AR. FFPE-FASP was tested on several samples, and differential analysis was used to compare the tissues of diverticulitis patients (healthy and inflammatory tissues). In this differential proteomic analysis using retrospective clinical FFPE material, FFPE-FASP was reproducible and provided a high number of confident protein identifications, highlighting potential protein biomarkers. BIOLOGICAL SIGNIFICANCE: In clinical proteomics, FFPE is an important resource for retrospective analysis and for the discovery of biomarkers. The challenge for FFPE shotgun proteomic analysis is preparation by an efficient and reproducible protocol, which includes protein extraction and digestion. In this study, we analyzed two different methods and evaluated their repeatabilities and efficiencies. We illustrated the reproducibility of the most efficient method, FFPE-FASP, by a pilot study on diverticulitis tissue and on FFPE samples amount accessible in hospital biobanks. These data showed that FFPE is suitable for use in clinical proteomics, especially when the FFPE-FASP method is combined with label-free shotgun proteomics as described in the workflow presented in this work.
Klockenbusch, C., O'Hara, J.E., Kast, J., Advancing formaldehyde cross-linking towards quantitative proteomic applications (2012) Anal Bioanal Chem, 404, pp. 1057-1067
Magdeldin, S., Yamamoto, T., Toward deciphering proteomes of formalin-fixed paraffin-embedded (FFPE) tissues (2012) Proteomics, 12, pp. 1045-1058
Ikeda, K., Monden, T., Kanoh, T., Tsujie, M., Izawa, H., Haba, A., Extraction and analysis of diagnostically useful proteins from formalin-fixed, paraffin-embedded tissue sections (1998) J Histochem Cytochem, 46, pp. 397-403
Gustafsson, J.O., Oehler, M.K., McColl, S.R., Hoffmann, P., Citric acid antigen retrieval (CAAR) for tryptic peptide imaging directly on archived formalin-fixed paraffin-embedded tissue (2010) J Proteome Res, 9, pp. 4315-4328
Fowler, C.B., Chesnick, I.E., Moore, C.D., O'Leary, T.J., Mason, J.T., Elevated pressure improves the extraction and identification of proteins recovered from formalin-fixed, paraffin-embedded tissue surrogates (2010) PLoS One, 5, p. e14253
Takadate, T., Onogawa, T., Fukuda, T., Motoi, F., Suzuki, T., Fujii, K., Novel prognostic protein markers of resectable pancreatic cancer identified by coupled shotgun and targeted proteomics using formalin-fixed paraffin-embedded tissues (2013) Int J Cancer, 132, pp. 1368-1382
Tanca, A., Addis, M.F., Simula, M.P., Pagnozzi, D., Biosa, G., Pisanu, S., Evaluation of the suitability of archival Bouin-fixed paraffin-embedded tissue specimens to proteomic investigation (2012) Electrophoresis, 33, pp. 1375-1384
Jain, M.R., Li, Q., Liu, T., Rinaggio, J., Ketkar, A., Tournier, V., Proteomic identification of immunoproteasome accumulation in formalin-fixed rodent spinal cords with experimental autoimmune encephalomyelitis (2012) J Proteome Res, 11, pp. 1791-1803
Maes, E., Broeckx, V., Mertens, I., Sagaert, X., Prenen, H., Landuyt, B., Analysis of the formalin-fixed paraffin-embedded tissue proteome: pitfalls, challenges, and future prospectives (2013) Amino Acids, 45, pp. 205-218
Fowler, C.B., O'Leary, T.J., Mason, J.T., Toward improving the proteomic analysis of formalin-fixed, paraffin-embedded tissue (2013) Expert Rev Proteomics, 10, pp. 389-400
Gustafsson, J.O., Eddes, J.S., Meding, S., Koudelka, T., Oehler, M.K., McColl, S.R., Internal calibrants allow high accuracy peptide matching between MALDI imaging MS and LC-MS/MS (2012) J Proteomics, 75, pp. 5093-5105
Wisniewski, J.R., Nagaraj, N., Zougman, A., Gnad, F., Mann, M., Brain phosphoproteome obtained by a FASP-based method reveals plasma membrane protein topology (2010) J Proteome Res, 9, pp. 3280-3289
Ostasiewicz, P., Zielinska, D.F., Mann, M., Wisniewski, J.R., Proteome, phosphoproteome, and N-glycoproteome are quantitatively preserved in formalin-fixed paraffin-embedded tissue and analyzable by high-resolution mass spectrometry (2010) J Proteome Res, 9, pp. 3688-3700
Wisniewski, J.R., Zielinska, D.F., Mann, M., Comparison of ultrafiltration units for proteomic and N-glycoproteomic analysis by the filter-aided sample preparation method (2011) Anal Biochem, 410, pp. 307-309
Balgley, B.M., Guo, T., Zhao, K., Fang, X., Tavassoli, F.A., Lee, C.S., Evaluation of archival time on shotgun proteomics of formalin-fixed and paraffin-embedded tissues (2009) J Proteome Res, 8, pp. 917-925
Maes, E., Valkenborg, D., Mertens, I., Broeckx, V., Baggerman, G., Sagaert, X., Proteomic analysis of formalin-fixed paraffin-embedded colorectal cancer tissue using tandem mass tag protein labeling (2013) Mol Biosyst, 9, pp. 2686-2695
Yang, H.Y., Chay, K.O., Kwon, J., Kwon, S.O., Park, Y.K., Lee, T.H., Comparative proteomic analysis of cysteine oxidation in colorectal cancer patients (2013) Mol Cells, 35, pp. 533-542
Silva, J.C., Denny, R., Dorschel, C.A., Gorenstein, M., Kass, I.J., Li, G.Z., Quantitative proteomic analysis by accurate mass retention time pairs (2005) Anal Chem, 77, pp. 2187-2200
Begcevic, I., Kosanam, H., Martinez-Morillo, E., Dimitromanolakis, A., Diamandis, P., Kuzmanov, U., Semiquantitative proteomic analysis of human hippocampal tissues from Alzheimer's disease and age-matched control brains (2013) Clin Proteomics, 10, p. 5
Cho, C.K., Smith, C.R., Diamandis, E.P., Amniotic fluid proteome analysis from Down syndrome pregnancies for biomarker discovery (2010) J Proteome Res, 9, pp. 3574-3582
John, D., Bancroft, M.G., (2007) Theory and practice of histological techniques, , Churchill Livingstone Elsevier
Silva, J.C., Gorenstein, M.V., Li, G.Z., Vissers, J.P., Geromanos, S.J., Absolute quantification of proteins by LCMSE: a virtue of parallel MS acquisition (2006) Mol Cell Proteomics, 5, pp. 144-156
Vissers, J.P., Langridge, J.I., Aerts, J.M., Analysis and quantification of diagnostic serum markers and protein signatures for Gaucher disease (2007) Mol Cell Proteomics, 6, pp. 755-766
Richardson, K., Denny, R., Hughes, C., Skilling, J., Sikora, J., Dadlez, M., A probabilistic framework for peptide and protein quantification from data-dependent and data-independent LC-MS proteomics experiments (2012) Omics J Integr Biol, 16, pp. 468-482
Richardson, K.J.D.R., Skilling, J., (2005) A mass spectrometer and a method of spectrometry are disclosed wherein liquid chromatography mass spectral data are probabilistically clustered on the basis of mass to charge ratio and retention time
Conesa, A., Gotz, S., Garcia-Gomez, J.M., Terol, J., Talon, M., Robles, M., Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research (2005) Bioinformatics, 21, pp. 3674-3676
Nirmalan, N.J., Hughes, C., Peng, J., McKenna, T., Langridge, J., Cairns, D.A., Initial development and validation of a novel extraction method for quantitative mining of the formalin-fixed, paraffin-embedded tissue proteome for biomarker investigations (2011) J Proteome Res, 10, pp. 896-906
Tanca, A., Pagnozzi, D., Burrai, G.P., Polinas, M., Uzzau, S., Antuofermo, E., Comparability of differential proteomics data generated from paired archival fresh-frozen and formalin-fixed samples by GeLC-MS/MS and spectral counting (2012) J Proteomics, 77, pp. 561-576
Jimenez, C.R., Knol, J.C., Meijer, G.A., Fijneman, R.J., Proteomics of colorectal cancer: overview of discovery studies and identification of commonly identified cancer-associated proteins and candidate CRC serum markers (2010) J Proteomics, 73, pp. 1873-1895
Goyette, J., Geczy, C.L., Inflammation-associated S100 proteins: new mechanisms that regulate function (2011) Amino Acids, 41, pp. 821-842
Pieters, J., Coronin 1 in innate immunity (2008) Subcell Biochem, 48, pp. 116-123
Pucci, S., Bonanno, E., Sesti, F., Mazzarelli, P., Mauriello, A., Ricci, F., Clusterin in stool: a new biomarker for colon cancer screening? (2009) Am J Gastroenterol, 104, pp. 2807-2815
Han, N.Y., Choi, W., Park, J.M., Kim, E.H., Lee, H., Hahm, K.B., Label-free quantification for discovering novel biomarkers in the diagnosis and assessment of disease activity in inflammatory bowel disease (2013) J Dig Dis, 14, pp. 166-174
