Premium
An in‐gel digestion procedure that facilitates the identification of highly hydrophobic proteins by electrospray ionization‐mass spectrometry analysis
Author(s) -
CastellanosSerra Lila,
Ramos Yassel,
Huerta Vivian
Publication year - 2005
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200401164
Subject(s) - chemistry , chromatography , mass spectrometry , electrospray ionization , bottom up proteomics , digestion (alchemy) , sample preparation in mass spectrometry , protein mass spectrometry , trypsin , electrospray , biochemistry , enzyme
A procedure is described for in‐gel tryptic digestion of proteins that allows the direct analysis of eluted peptides in electrospray ionization (ESI) mass spectrometers without the need of a postdigestion desalting step. It is based on the following principles: (a) a thorough desalting of the protein in‐gel before digestion that takes advantage of the excellent properties of acrylamide polymers for size exclusion separations, (b) exploiting the activity of trypsin in water, in the absence of inorganic buffers, and (c) a procedure for peptide extraction using solvents of proven efficacy with highly hydrophobic peptides. Quality of spectra and sequence coverage are equivalent to those obtained after digestion in ammonium bicarbonate for hydrophilic proteins detected with Coomassie blue, mass spectrometry‐compatible silver or imidazole‐zinc but are significantly superior for highly hydrophobic proteins, such as membrane proteins with several transmembrane domains. ATPase subunit 9 (GRAVY 1.446) is a membrane protein channel, lipid‐binding protein for which both the conventional in‐gel digestion protocol and in solution digestion failed. It was identified with very high sequence coverage. Sample handling after digestion is notably simplified as peptides are directly loaded into the ESI source without postdigestion processing, increasing the chances for the identification of hydrophobic peptides.