Abstract
Purification and identification of candidate biomarkers is a critical step in the biomarker development process, since it provides insight into the disease biology and facilitates the development of analyte-specific assays. Top-down biomarker discovery workflows like SELDI-TOF MS yield candidate markers that are identified based on native mass. Positive identification of these candidate biomarkers requires further enrichment and/or purification. While purification methods must be optimized for each protein target, there are two general workflows. Native peptides under approximately 4 kDa can be subjected to direct sequence analysis using a tandem mass spectrometer whereas proteins over approximately 4 kDa usually require proteolytic digestion prior to MS/MS analysis. In both cases, partial purification is usually necessary to enrich the candidate biomarker relative to other proteins in a complex biological mixture. This chapter provides detailed protocols for protein purification (including anion exchange, metal affinity, and reverse phase chromatography as well as SDS-PAGE) and identification (including protein processing, digestion, and database searching).
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References
Peng, J., Stanley, A.J., Cairns, D., Selby, P.J., Banks, R.E.(2009) Using the protein chip interface with quadrupole time-of-flight mass spectrometry to directly identify peaks in SELDI profiles – initial evaluation using low molecular weight serum peaks. Proteomics 9, 492–498.
Miguet, L., Bogumil, R., Decloquement, P., Herbrecht, R., Potier, N., Mauvieux, L., Van Dorsselaer, A. (2006) Discovery and identification of potential biomarkers in a prospective study of chronic lymphoid malignancies using SELDI-TOF-MS. J Proteome Res 5, 2258–2269.
Brown, A.G., Leite, R.S., Engler, A.J., Discher, D.E., Strauss, J.F. (2006) A hemoglobin fragment found in cervicovaginal fluid from women in labor potentiates the action of agents that promote contraction of smooth muscle cells. Peptides 27, 1794–1800.
Simonsen, A.H., McGuire, J, Podust, V.N., Davies, H., Minthon, L., Skoog, I., Andreasen, N., Wallin, A., Waldemar, G., Blennow, K. (2008) Identification of a novel panel of cerebrospinal fluid biomarkers for Alzheimer’s disease. Neurobiol Aging. 29, 961–968.
Ye, B., Skates, S., Mok, S.C., Horick, N.K., Rosenberg, H.F., Vitonis, A., Edwards, D., Sluss, P., Han, W.K., Berkowitz, R.S., Cramer, D.W. (2006) Proteomic-based discovery and characterization of glycosylated eosinophil-derived neurotoxin and COOH-terminal osteopontin fragments for ovarian cancer in urine. Clin Cancer Res 12, 432–441.
Cho, WC., Yip, T.T., Yip, C., Yip, V., Thulasiraman, V., Ngan, R.K., Yip, T.T., Lau, W.H., Au, J.S., Law, S.C., Cheng, W.W., Ma, V.W., Lim, C.K. (2004) Identification of serum amyloid A protein as a potentially useful biomarker to monitor relapse of nasopharyngeal cancer by serum proteomic profiling. Clin Cancer Res 10, 43–52.
Zhang, Z., Bast, R.C. Jr., Yu, Y., Li, J., Sokoll, L.J., Rai, A.J., Rosenzweig, J.M., Cameron, B., Wang, Y.Y., Meng, X.Y., Berchuck, A., Van Haaften-Day, C., Hacker, N.F., de Bruijn, H.W., van der Zee, A.G., Jacobs, I.J., Fung, E.T., Chan, D.W.(2004) Three biomarkers identified from serum proteomic analysis for the detection of early stage ovarian cancer. Cancer Res 64, 5882–5890.
Vermeulen, R., Lan, Q., Zhang, L., Gunn, L., McCarthy, D., Woodbury, R.L., McGuire, M., Podust, V.N., Li, G., Chatterjee, N., Mu, R., Yin, S., Rothman, N., Smith, M.T.(2005) Decreased levels of CXC-chemokines in serum of benzene-exposed workers identified by array-based proteomics. Proc Natl Acad Sci 102, 17041–17046.
Huang, J.T., Leweke, F.M., Oxley, D., Wang, L., Harris, N., Koethe, D., Gerth, C.W., Nolden, B.M., Gross, S., Schreiber, D., Reed, B., Bahn, S. (2006) Disease biomarkers in cerebrospinal fluid of patients with first-onset psychosis, PLoS Med 3, 2145–2158.
Melle, C., Bogumil, R., Ernst, G., Schimmel, B., Bleul, A., von Eggeling, F. (2006) Detection and identification of heat shock protein 10 as a biomarker in colorectal cancer by protein profiling. Proteomics 6, 2600–2608.
Nguyen, M.T., Dent, C.L., Ross, G.F., Harris, N., Manning, P.B., Mitsnefes, M.M., Devarajan, P.(2008) Urinary aprotinin as a predictor of acute kidney injury after cardiac surgery in children receiving aprotinin therapy. Pediatr Nephrol 23, 1317–1326.
Currid, C.A., O’Connor, D.P., Chang, B.D., Gebus, C., Harris, N., Dawson, K.A., Dunn, M.J., Pennington, S.R., Roninson, I.B., Gallagher, W.M. (2006) Proteomic analysis of factors released from p21-overexpressing tumour cells. Proteomics 6, 3739–3753.
Guerrier, L., Righetti, P.G., Boschetti, E. (2008) Reduction of dynamic protein concentration range of biological extracts for the discovery of low-abundance proteins by means of hexapeptide ligand library. Nat Protoc 3, 883–890.
Sihlbom, C., Kanmert, I., Bahr, H., Davidsson, P. (2008) Evaluation of the combination of bead technology with SELDI-TOF-MS and 2-D DIGE for detection of plasma proteins. J Proteome Res 7, 4191–4198.
Boschetti, E., Righetti, P.G. (2008) The ProteoMiner in the proteomic arena: a non-depleting tool for discovering low-abundance species. J Proteomics 71, 255–64.
Sennels, L., Salek, M., Lomas, L., Boschetti, E., Righetti, P.G., Rappsilber, J. (2007) Proteomic analysis of human blood serum using peptide library beads. J Proteome Res 6, 4055–4062.
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Bulman, A.L., Dalmasso, E.A. (2012). Purification and Identification of Candidate Biomarkers Discovered Using SELDI-TOF MS. In: Clarke, C., McCarthy, D. (eds) SELDI-TOF Mass Spectrometry. Methods in Molecular Biology, vol 818. Springer, New York, NY. https://doi.org/10.1007/978-1-61779-418-6_4
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DOI: https://doi.org/10.1007/978-1-61779-418-6_4
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