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Cancer Signaling Network Analysis by Quantitative Mass Spectrometry

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Targeted Therapies

Part of the book series: Molecular and Translational Medicine ((MOLEMED))

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Abstract

Global analysis of protein phosphorylation by mass spectrometry (MS) provides unbiased, discovery-based, site-specific monitoring of phosphorylation sites governing cell signaling networks involved in cancer progression and therapeutic resistance. In this chapter, advances in MS instrumentation and methodology for the identification and quantification of protein phosphorylation are discussed. These topics include (1) advantages and limitations of current MS-based protocols, (2) fundamentals of phosphopeptide fragmentation and identification, (3) selection of MS instrumentation, (4) fractionation and enrichment methods for detecting phosphorylated proteins/peptides, and (5) methods for phosphoproteome quantification. The final two topics represent the most important subjects of this chapter, as fractionation, enrichment, and quantification are crucially important to the generation of high quality MS-based phosphoproteomic data. These sections detail the use of immunoaffinity enrichment, immobilized metal affinity chromatography (IMAC), metal oxide affinity chromatography (MOAC), and strong cation exchange (SCX) chromatography as key methods for enriching and fractionating complex biological samples for phosphoproteomic analysis. Quantification of changes in signaling networks at the phosphoproteomic level through metabolic labeling (e.g., SILAC), chemical modification (e.g., iTRAQ), or label-free quantification is presented. As significant progress in detection and quantification strategies in phosphoproteomic research has arisen over the last decade, implementation of these approaches will enhance our understanding of cell signaling networks involved in cancer progression and thereby improve therapeutic targeting and monitoring of therapeutic efficacy.

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Abbreviations

ESI:

Electrospray ionization

IMAC:

Immobilized metal affinity chromatography

LC-MS/MS:

Liquid chromatography tandem mass spectrometry

MALDI:

Matrix-assisted laser desorption ionization

MOAC:

Metal oxide affinity chromatography

SCX:

Strong cation exchange

SILAC:

Stable isotope labeling by amino acids in cell culture

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Acknowledgments

We thank Amanda Del Rosario and other members of the White laboratory for helpful discussions regarding the contents of this chapter. This work was supported by a Genetech Postdoctoral Fellowship (to J.R.N.) and by National Cancer Institute (NCI) Grants R01-CA118705 (to F.M.W.).

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  1. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA

    Forest M. White

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  1. Jason R. Neil
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Correspondence to Forest M. White .

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  1. Health System, Deptartment of Microbiology and Cancer C, University of Virginia, Charlottesville, 22908-0734, Virgin Islands, USA

    Daniel Gioeli

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Neil, J.R., White, F.M. (2011). Cancer Signaling Network Analysis by Quantitative Mass Spectrometry. In: Gioeli, D. (eds) Targeted Therapies. Molecular and Translational Medicine. Humana Press. https://doi.org/10.1007/978-1-60761-478-4_3

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