title: Full Membrane Protein Coverage Digestion and Quantitative Bottom-Up Mass Spectrometry Proteomics.
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.
authors.affiliation: Department of Pharmacology, David Geffen School of Medicine, UCLA, Los Angeles, CA, 90095, USA.
authors.forename: Joseph
authors.initials: J
authors.lastname: Capri
authors.affiliation: The Pasarow Mass Spectrometry Laboratory, The Jane and Terry Semel Institute for Neuroscience and Human Behavior, David Geffen School of Medicine, UCLA, Box 42, 760 Westwood Plaza, Los Angeles, CA, 90095, USA. jpw@chem.ucla.edu.
authors.forename: Julian P
authors.initials: JP
authors.lastname: Whitelegge
mesh_terms: D006868:Hydrolysis; D013058:Mass Spectrometry / Q000379:methods; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D008745:Methylation; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D059748:Proteolysis; D020543:Proteome; D040901:Proteomics / Q000379:methods; D014357:Trypsin / Q000378:metabolism
chemical_list: D008565:Membrane Proteins; D010455:Peptides; D020543:Proteome; D014357:Trypsin
keywords: Dimethylation; Electrospray ionization; Phase transfer; Proteome; StageTip; Trypsin