Chapter title |
Full Membrane Protein Coverage Digestion and Quantitative Bottom-Up Mass Spectrometry Proteomics
|
---|---|
Chapter number | 6 |
Book title |
Proteomics
|
Published in |
Methods in molecular biology, February 2017
|
DOI | 10.1007/978-1-4939-6747-6_6 |
Pubmed ID | |
Book ISBNs |
978-1-4939-6745-2, 978-1-4939-6747-6
|
Authors |
Joseph Capri, Julian P. Whitelegge Ph.D., Julian P. Whitelegge |
Editors |
Lucio Comai, Jonathan E. Katz, Parag Mallick |
Abstract |
A true and accurate bottom-up global proteomic measurement will only be achieved when all proteins in a sample can be digested efficiently and at least some peptides recovered on which to base an estimate of abundance. Integral membrane proteins make up around one-third of the proteome and require specialized protocols if they are to be successfully solubilized for efficient digestion by the enzymes used in bottom-up proteomics. The protocol described relies upon solubilization using the detergents sodium deoxycholate and lauryl sarcosine with heating to 95 °C. A subset of peptides is purified by reverse-phase solid-phase extraction and fractionated by strong-cation exchange prior to nano-liquid chromatography with data-dependent tandem mass spectrometry. For quantitative proteomics experiments a protocol is described for stable-isotope coding of peptides using dimethylation of primary amines allowing for three-way sample multiplexing. |
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