Abstract
In this chapter we describe a method to analyze human serum with the goal of discovering disease-related changes in the serum proteome. The methodology is based on the removal of the six most abundant serum proteins by immunoaffinity chromatography. This step is followed by trypsin digestion and reversed-phase high-performance liquid chromatography (HPLC) coupled on-line to mass spectrometry (MS) using either a capillary HPLC or a microfluidics chip HPLC system. The obtained, highly complex data sets are processed and statistically analyzed to discover significant differences between groups of samples. The complete analytical procedure will be described with serum samples, to which a given amount of horse heart cytochrome c has been added as well as with serum samples from early stage cervical cancer patients prior to and after therapy. The use of reversed-phase HPLC to separate serum proteins at 80°C with subsequent analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in order to lower the concentration sensitivity will also be briefly described.
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References
Villanueva, J., Philip, J., Chaparro, C. A., Li, Y., Toledo-Crow, R., DeNoyer, L., Fleisher, M., Robbins, R. J., and Tempst, P. (2005) Correcting common errors in identifying cancer-specific serum peptide signatures. J. Proteome Res. 4, 1060–1072.
Kemperman, R. F., Horvatovich, P. L., Hoekman, B., Reijmers, T. H., Muskiet, F. A., and Bischoff, R. (2007) Comparative urine analysis by liquid chromatography-mass spectrometry and multivariate statistics: method development, evaluation, and application to proteinuria. J. Proteome Res. 6, 194–206.
Govorukhina, N. I., Reijmers, T. H., Nyangoma, S. O., van der Zee, A. G. J., Jansen, R. C., and Bischoff, R. (2006) Analysis of human serum by LC-MS: improved sample preparation and data analysis. J. Chromatogr. A 110, 142–150.
Dekker, L. J., Bosman, J., Burgers, P. C., van Rijswijk, A., Freije, R., Luider, T., and Bischoff, R. (2007) Depletion of high-abundance proteins from serum by immunoaffinity chromatography: a MALDI-FT-MS study. J. Chromatogr. B 847, 65–69.
Martosella, J., Zolotarjova, N., Liu, H., Nicol, G., and Boyes, B. E. (2005) Reversed-phase high-performance liquid chromatographic prefractionation of immunodepleted human serum proteins to enhance mass spectrometry identification of lower-abundant proteins. J. Proteome Res. 4, 1522–1537.
Horvatovich, P., Govorukhina, N. I., Reijmers, T. H., van der Zee, A. G. J., and Bischoff, R. (2007) Evaluation of HPLC-chip/MS platform for label-free profiling for biomarker discovery, Electrophoresis 28, 4493–4505.
Tibshirani, R., Hastie, T., Narasimhan, B., and Chu, G. (2002) Diagnosis of multiple cancer types by shrunken centroids of gene expression. Proc. Natl. Acad. Sci. USA 99, 6567–6572.
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© 2008 Humana Press, Totowa, NJ
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Govorukhina, N., Horvatovich, P., Bischoff, R. (2008). Label-Free Proteomics of Serum. In: Thompson, J.D., Ueffing, M., Schaeffer-Reiss, C. (eds) Functional Proteomics. Methods in Molecular Biology, vol 484. Humana Press. https://doi.org/10.1007/978-1-59745-398-1_5
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DOI: https://doi.org/10.1007/978-1-59745-398-1_5
Publisher Name: Humana Press
Print ISBN: 978-1-58829-971-0
Online ISBN: 978-1-59745-398-1
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