Chapter title |
Determining the Composition and Stability of Protein Complexes Using an Integrated Label-Free and Stable Isotope Labeling Strategy.
|
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Chapter number | 3 |
Book title |
Quantitative Proteomics by Mass Spectrometry
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Published in |
Methods in molecular biology, January 2016
|
DOI | 10.1007/978-1-4939-3524-6_3 |
Pubmed ID | |
Book ISBNs |
978-1-4939-3522-2, 978-1-4939-3524-6
|
Authors |
Todd M. Greco, Amanda J. Guise, Ileana M. Cristea |
Editors |
Salvatore Sechi |
Abstract |
In biological systems, proteins catalyze the fundamental reactions that underlie all cellular functions, including metabolic processes and cell survival and death pathways. These biochemical reactions are rarely accomplished alone. Rather, they involve a concerted effect from many proteins that may operate in a directed signaling pathway and/or may physically associate in a complex to achieve a specific enzymatic activity. Therefore, defining the composition and regulation of protein complexes is critical for understanding cellular functions. In this chapter, we describe an approach that uses quantitative mass spectrometry (MS) to assess the specificity and the relative stability of protein interactions. Isolation of protein complexes from mammalian cells is performed by rapid immunoaffinity purification, and followed by in-solution digestion and high-resolution mass spectrometry analysis. We employ complementary quantitative MS workflows to assess the specificity of protein interactions using label-free MS and statistical analysis, and the relative stability of the interactions using a metabolic labeling technique. For each candidate protein interaction, scores from the two workflows can be correlated to minimize nonspecific background and profile protein complex composition and relative stability. |
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Unknown | 33 | 100% |
Demographic breakdown
Readers by professional status | Count | As % |
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Student > Bachelor | 7 | 21% |
Researcher | 6 | 18% |
Unspecified | 3 | 9% |
Student > Ph. D. Student | 3 | 9% |
Other | 2 | 6% |
Other | 6 | 18% |
Unknown | 6 | 18% |
Readers by discipline | Count | As % |
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Pharmacology, Toxicology and Pharmaceutical Science | 1 | 3% |
Other | 5 | 15% |
Unknown | 7 | 21% |