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High Throughput Profiling of Serum Phosphoproteins/Peptides Using the SELDI-TOF-MS Platform

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Book cover SELDI-TOF Mass Spectrometry

Part of the book series: Methods in Molecular Biology ((MIMB,volume 818))

Abstract

Protein phosphorylation is a dynamic post-translational modification that plays a critical role in the regulation of a wide spectrum of biological events and cellular functions including signal transduction, gene expression, cell proliferation, and apoptosis. Determination of the sites and magnitudes of protein phosphorylation has been an essential step in the analysis of the control of many biological systems. A high throughput analysis of phosphorylation of proteins would provide a simple, logical, and useful tool for a functional dissection and prediction of biological functions and signaling pathways in association with these important molecular events. We have developed a functional proteomics technique using the ProteinChip array-based SELDI-TOF-MS analysis for high throughput profiling of phosphoproteins/phosphopeptides in human serum for the early detection and diagnosis as well as for the molecular staging of human cancer. The methodology and experimental approach consists of five steps: (1) generation of a total peptide pool of serum proteins by a global trypsin digestion; (2) rapid isolation of phosphopeptides from the total serum peptide pool by an affinity selection, purification, and enrichment using a novel automated micro-bioprocessing system with phospho-antibody-conjugated paramagnetic beads and a hybrid magnet plate; (3) high throughput phosphopeptide analysis on ProteinChip arrays by automated SELDI-TOF-MS; and (4) bioinformatics and statistical methods for data analysis. This method with appropriate modifications may be equally applicable to serine-, threonine- and tyrosine-phosphorylated proteins and for selectively isolating, profiling, and identifying phosphopeptides present in a highly complex phosphor-peptide mixture prepared from various human specimens such as cells, tissue samples, and serum and other body fluids.

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Acknowledgments

The authors would like to thank Drs. Xifeng Wu and Margaret Spitz for providing lung cancer serum samples for serum phosphopeptide profiling and analysis and Dr. Kevin Coombes for bioinformatics and statistical analysis, at The University of Texas M. D. Anderson Cancer Center, Houston, TX, and David Humphries at Lawrence Berkeley National Laboratory, Oak Land, CA for developing hybrid magnetic plates for serum phosphopeptide enrichment. This work was partially supported by grants from NIH/NCI SPORE P50CA070907, RO1CA116322, and MMHCC U01CA105352; DOD PROSPECT W81XWH-0710306; The University of Texas M. D. Anderson Cancer Center Support Core Grant (CA16672).

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Authors and Affiliations

  1. Department of Thoracic and Cardiovascular Surgery, The University of Texas, Anderson Cancer Center, Houston, TX, USA

    Lin Ji, Gitanjali Jayachandran & Jack A. Roth

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  1. Lin Ji
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  2. Gitanjali Jayachandran
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  3. Jack A. Roth
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Correspondence to Lin Ji .

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Editors and Affiliations

  1. The University of Texas, Department of Translational Research, M.D. Anderson Cancer Center, Holcombe Blvd. 1515, Houston, 77030-3722, Texas, USA

    Charlotte H. Clarke

  2. Bio-Rad Laboratories, Great Valley Pkwy. 277, Malvern, 19355, Pennsylvania, USA

    Diane L. Bankert McCarthy

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Ji, L., Jayachandran, G., Roth, J.A. (2012). High Throughput Profiling of Serum Phosphoproteins/Peptides Using the SELDI-TOF-MS Platform. 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_14

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  • DOI: https://doi.org/10.1007/978-1-61779-418-6_14

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  • Print ISBN: 978-1-61779-417-9

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