Abstract
In-depth quantitative profiling of human plasma samples for biomarker discovery remains quite challenging. One promising alternative to chemical derivatization with stable isotope labels for quantitative comparisons is direct, label-free, quantitative comparison of raw LC–MS data. But, in order to achieve high-sensitivity detection of low-abundance proteins, plasma proteins must be extensively pre-fractionated, and results from LC–MS runs of all fractions must be integrated efficiently in order to avoid misidentification of variations in fractionation from sample to sample as “apparent” biomarkers. This protocol describes a powerful 3D protein profiling method for comprehensive analysis of human serum or plasma proteomes, which combines abundant protein depletion and high-sensitivity GeLC–MS/MS with label-free quantitation of candidate biomarkers.
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References
Jacobs, J. M., Adkins, J. N., Qian, W. J., et al. (2005) Utilizing human blood plasma for proteomic biomarker discovery, J Proteome Res 4, 1073–1085.
Anderson, N. L., and Anderson, N. G. (2002) The human plasma proteome: history, character, and diagnostic prospects, Mol Cell Proteomics 1, 845–867.
Hanash, S. M., Pitteri, S. J., and Faca, V. M. (2008) Mining the plasma proteome for cancer biomarkers, Nature 452, 571–579.
Hu, S., Loo, J. A., and Wong, D. T. (2006) Human body fluid proteome analysis, Proteomics 6, 6326–6353.
Hoffman, S. A., Joo, W. A., Echan, L. A., and Speicher, D. W. (2007) Higher dimensional (Hi-D) separation strategies dramatically improve the potential for cancer biomarker detection in serum and plasma, J Chromatogr B Analyt Technol Biomed Life Sci 849, 43–52.
Omenn, G. S., States, D. J., Adamski, M., et al. (2005) Overview of the HUPO Plasma Proteome Project: results from the pilot phase with 35 collaborating laboratories and multiple analytical groups, generating a core dataset of 3020 proteins and a publicly-available database, Proteomics 5, 3226–3245.
States, D. J., Omenn, G. S., Blackwell, T. W., et al. (2006) Challenges in deriving high-confidence protein identifications from data gathered by a HUPO plasma proteome collaborative study, Nat Biotechnol 24, 333–338.
Tirumalai, R. S., Chan, K. C., Prieto, D., et al. (2003) Characterization of the low molecular weight human serum proteome, Mol Cell Proteomics 2, 1096–1103.
America, A. H., and Cordewener, J. H. (2008) Comparative LC-MS: a landscape of peaks and valleys, Proteomics 8, 731–749.
Bantscheff, M., Schirle, M., Sweetman, G., Rick, J., and Kuster, B. (2007) Quantitative mass spectrometry in proteomics: a critical review, Anal Bioanal Chem 389, 1017–1031.
Faca, V., Pitteri, S. J., Newcomb, L., et al. (2007) Contribution of protein fractionation to depth of analysis of the serum and plasma proteomes, J Proteome Res 6, 3558–3565.
Lee, H. J., Lee, E. Y., Kwon, M. S., and Paik, Y. K. (2006) Biomarker discovery from the plasma proteome using multidimensional fractionation proteomics, Curr Opin Chem Biol 10, 42–49.
Pernemalm, M., Orre, L. M., Lengqvist, J., et al. (2008) Evaluation of three principally different intact protein prefractionation methods for plasma biomarker discovery, J Proteome Res 7, 2712–2722.
Echan, L. A., Tang, H. Y., Ali-Khan, N., et al. (2005) Depletion of multiple high-abundance proteins improves protein profiling capacities of human serum and plasma, Proteomics 5, 3292–3303.
Pieper, R., Su, Q., Gatlin, C. L., et al. (2003) Multi-component immunoaffinity subtraction chromatography: an innovative step towards a comprehensive survey of the human plasma proteome, Proteomics 3, 422–432.
Qian, W. J., Kaleta, D. T., Petritis, B. O., et al. (2008) Enhanced detection of low abundance human plasma proteins using a tandem IgY12-SuperMix immunoaffinity separation strategy, Mol Cell Proteomics 7, 1963–1973.
Steen, H., and Mann, M. (2004) The ABC’s (and XYZ’s) of peptide sequencing, Nat Rev Mol Cell Biol 5, 699–711.
Schirle, M., Heurtier, M. A., and Kuster, B. (2003) Profiling core proteomes of human cell lines by one-dimensional PAGE and liquid chromatography-tandem mass spectrometry, Mol Cell Proteomics 2, 1297–1305.
Old, W. M., Meyer-Arendt, K., Aveline-Wolf., et al. (2005) Comparison of label-free methods for quantifying human proteins by shotgun proteomics, Mol Cell Proteomics 4, 1487–1502.
Mann, M., and Kelleher, N. L. (2008) Precision proteomics: the case for high resolution and high mass accuracy, Proc Natl Acad Sci U S A 105, 18132–18138.
Echan, L. A., and Speicher, D. W. (2009) Immunoaffinity depletion of high abundance plasma and serum proteins, In The Protein Protocols Handbook (Walker, J. M., Ed.) 2nd ed., pp 139-153, Humana Press, New York.
Rai, A. J., Gelfand, C. A., Haywood, B. C., et al. (2005) HUPO Plasma Proteome Project specimen collection and handling: towards the standardization of parameters for plasma proteome samples, Proteomics 5, 3262–3277.
Sadiq, S. T., and Agranoff, D. (2008) Pooling serum samples may lead to loss of potential biomarkers in SELDI-ToF MS proteomic profiling, Proteome Sci 6, 16.
Karp, N. A., and Lilley, K. S. (2009) Investigating sample pooling strategies for DIGE experiments to address biological variability, Proteomics 9, 388–397.
Peng, X., Wood, C. L., Blalock, E. M., Chen, K. C., Landfield, P. W., and Stromberg, A. J. (2003) Statistical implications of pooling RNA samples for microarray experiments, BMC Bioinformatics 4, 26.
Neubert, H., Bonnert, T. P., Rumpel, K., et al. (2008) Label-free detection of differential protein expression by LC/MALDI mass spectrometry, J Proteome Res 7, 2270–2279.
Acknowledgments
This work was supported by NIH grants HD036455 (K.T.B.), CA120393 and CA131582 (D.W.S.), and by an institutional grant to the Wistar Institute (NCI Cancer Core Grant CA10815).
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Beer, L.A., Tang, HY., Barnhart, K.T., Speicher, D.W. (2011). Plasma Biomarker Discovery Using 3D Protein Profiling Coupled with Label-Free Quantitation. In: Simpson, R., Greening, D. (eds) Serum/Plasma Proteomics. Methods in Molecular Biology, vol 728. Humana Press. https://doi.org/10.1007/978-1-61779-068-3_1
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DOI: https://doi.org/10.1007/978-1-61779-068-3_1
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