Premium
Trypsin digestion of proteins on intact immobilized pH gradient strips for surface matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometric analysis
Author(s) -
Iyer Srinivas,
Olivares José
Publication year - 2003
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.1199
Subject(s) - chemistry , chromatography , trypsin , mass spectrometry , matrix assisted laser desorption/ionization , immobilized ph gradient , desorption , matrix (chemical analysis) , sample preparation in mass spectrometry , electrophoresis , time of flight mass spectrometry , sample preparation , analytical chemistry (journal) , ionization , electrospray ionization , biochemistry , isoelectric focusing , enzyme , ion , organic chemistry , adsorption
Isolelectric focusing (IEF) of proteins on immobilized pH gradient (IPG) strips is an integral part of two‐dimensional (2D) electrophoresis‐based proteomics. Proteins can be effectively analyzed by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOFMS) on the intact strip itself, leading to the creation of a virtual 2D map giving pI and MW information, bypassing the second dimension SDS‐PAGE. Further, trypsin digestion of proteins on the strip can significantly aid the identification of IPG‐separated proteins. However, the small size of the peptides leads to diffusion along and outside the gel matrix. In this study, we describe a simple spray‐based procedure to perform ‘on‐strip’ trypsin digestion of proteins embedded in IPG strips. Examination of intact myoglobin and its tryptic peptides shows that post‐digestion diffusion of tryptic peptides is significantly minimized using this approach. Copyright © 2003 John Wiley & Sons, Ltd.