Abstract
To understand the biological processes in health and disease, it would be useful to profile all the nucleic acids, glycans, lipids, proteins, and metabolites in a particular system. Unfortunately, no single analytical approach exists that is capable of analyzing all of these elements simultaneously. Rather, each unique sample type requires distinct methods for preparation and analysis: genomics for nucleic acids, glycomics for glycans, lipidomics for lipids, proteomics for proteins, and metabolomics for metabolites. In proteomic analyses, unique experimental designs frequently apply various degrees of fractionation, diverse gel-based or gel-free separation techniques, and a host of mass spectrometric analytical approaches. These issues are discussed in the introduction to encourage the broad community of scientists to develop a specific method for each unique experiment. At the same time, this chapter describes several special sample preparation methods for LC-MS/MS using electrospray ionization and a linear ion-trap mass spectrometer. The methods are optimized, reproducible, and as simple as possible, and they are capable of generating excellent results. However, optimization for each unique experiment is always a good idea.
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Bodzon-Kulakowska, A., Bierczynska-Krzysik, A., Dylag, T., Drabik, A., Suder, P., Noga, M., Jarzebinska, J., and Silberring, J. (2007). Methods for samples preparation in proteomic research. J Chromatogr B Analyt Technol Biomed Life Sci 849, 1–31.
Chaiyarit, S., and Thongboonkerd, V. (2009). Comparative analyses of cell disruption methods for mitochondrial isolation in high-throughput proteomics study. Anal Biochem 394, 249–258.
Delom, F., and Chevet, E. (2006). Phosphoprotein analysis: From proteins to proteomes. Proteome Sci 4, 15.
Dormeyer, W., van Hoof, D., Mummery, C.L., Krijgsveld, J., and Heck, A.J. (2008). A practical guide for the identification of membrane and plasma membrane proteins in human embryonic stem cells and human embryonal carcinoma cells. Proteomics 8, 4036–4053.
Du, J., Xu, H., Wei, N., Wakim, B., Halligan, B., Pritchard, K.A., Jr., and Shi, Y. (2009). Identification of proteins interacting with GTP cyclohydrolase I. Biochem Biophys Res Commun 385, 143–147.
Essader, A.S., Cargile, B.J., Bundy, J.L., and Stephenson, J.L., Jr. (2005). A comparison of immobilized pH gradient isoelectric focusing and strong-cation-exchange chromatography as a first dimension in shotgun proteomics. Proteomics 5, 24–34.
Geng, M., Zhang, X., Bina, M., and Regnier, F. (2001). Proteomics of glycoproteins based on affinity selection of glycopeptides from tryptic digests. J Chromatogr B Biomed Sci Appl 752, 293–306.
Hale, J.E., Butler, J.P., Gelfanova, V., You, J.S., and Knierman, M.D. (2004). A simplified procedure for the reduction and alkylation of cysteine residues in proteins prior to proteolytic digestion and mass spectral analysis. Anal Biochem 333, 174–181.
Huynh, M.L., Russell, P., and Walsh, B. (2009). Tryptic digestion of in-gel proteins for mass spectrometry analysis. Methods Mol Biol 519, 507–513.
Keller, A., Nesvizhskii, A.I., Kolker, E., and Aebersold, R. (2002). Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Anal Chem 74, 5383–5392.
Lai, X.Y., Bacalla, R.L., Blazer-Yost, B.L., Hong, D., Mason, S.B., and Witzmann, F.A. (2008). Characterization of the renal cyst fluid proteome in autosomal dominant polycystic kidney disease (ADPKD) patients. Proteomics Clin Appl 2, 1140–1152.
Lai, X., Liangpunsakul, S., Crabb, D.W., Ringham, H.N., and Witzmann, F.A. (2009a). A proteomic workflow for discovery of serum carrier protein-bound biomarker candidates of alcohol abuse using LC-MS/MS. Electrophoresis 30, 2207–2214.
Lai, X.Y., Blazer-Yost, B.L., Gattone, V.H., Muchatuta, M.N., and Witzmann, F.A. (2009b). Protein composition of liver cyst fluid from the BALB/c-cpk/+ mouse model of autosomal recessive polycystic kidney disease. Proteomics 9, 3775–3782.
Lambert, J.P., Ethier, M., Smith, J.C., and Figeys, D. (2005). Proteomics: From gel based to gel free. Anal Chem 77, 3771–3787.
Lecchi, P., Gupte, A.R., Perez, R.E., Stockert, L.V., and Abramson, F.P. (2003). Size-exclusion chromatography in multidimensional separation schemes for proteome analysis. J Biochem Biophys Methods 56, 141–152.
Lipton, M.S., and Pasa-Tolic, L. (2009). Mass spectrometry of proteins and peptides: Methods and protocols. Methods Mol Biol 492, 1–474.
Nesvizhskii, A.I., Keller, A., Kolker, E., and Aebersold, R. (2003). A statistical model for identifying proteins by tandem mass spectrometry. Anal Chem 75, 4646–4658.
Pernemalm, M., Lewensohn, R., and Lehtio, J. (2009). Affinity prefractionation for MS-based plasma proteomics. Proteomics 9, 1420–1427.
Prescher, J.A., and Bertozzi, C.R. (2005). Chemistry in living systems. Nat Chem Biol 1, 13–21.
Richardson, M.R., Lai, X., Dixon, J.L., Sturek, M., and Witzmann, F.A. (2009). Diabetic dyslipidemia and exercise alter the plasma low-density lipoproteome in Yucatan pigs. Proteomics 9, 2468–2483.
Takatalo, M.S., Kouvonen, P., Corthals, G., Nyman, T.A., and Ronnholm, R.H. (2006). Identification of new Golgi complex specific proteins by direct organelle proteomic analysis. Proteomics 6, 3502–3508.
Wilkins, M.R., Pasquali, C., Appel, R.D., Ou, K., Golaz, O., Sanchez, J.C., Yan, J.X., Gooley, A.A., Hughes, G., Humphery-Smith, I., et al. (1996). From proteins to proteomes: Large scale protein identification by two-dimensional electrophoresis and amino acid analysis. Biotechnology 14, 61–65.
Witzmann, F.A. (2005). Preparation of Mammalian Tissue Samples for Two-Dimensional Electrophoresis. In The Proteomics Protocols Handbook, J.M. Walker, ed. (Totowa, NJ, Humana Press), pp. 31–35.
Witzmann, F.A., Clack, J.W., Geiss, K., Hussain, S., Juhl, M.J., Rice, C.M., and Wang, C. (2002). Proteomic evaluation of cell preparation methods in primary hepatocyte cell culture. Electrophoresis 23, 2223–2232.
Acknowledgments
The authors gratefully acknowledge the technical assistance of Mrs. Heather Ringham. Much of this work was supported by NIH-NIAAA R21 AA016-217-01 and AFOSR Grant FA9550-06-1-0083.
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Lai, X., Witzmann, F.A. (2011). Gel-Based and Gel-Free Sample Preparation for LC-MS/MS Analysis. In: Ivanov, A., Lazarev, A. (eds) Sample Preparation in Biological Mass Spectrometry. Springer, Dordrecht. https://doi.org/10.1007/978-94-007-0828-0_1
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DOI: https://doi.org/10.1007/978-94-007-0828-0_1
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