Trypsin digestion of proteins on intact immobilized pH gradient strips for surface matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis.
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.
Iyer
Srinivas
S
Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM 87545, USA.
Olivares
José
J
D000595:Amino Acid Sequence; D000818:Animals; D015180:Electrophoresis, Gel, Two-Dimensional; D006736:Horses; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D009206:Myocardium; D009211:Myoglobin / Q000737:chemistry / Q000378:metabolism; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000295:instrumentation / Q000379:methods*; D013552:Swine; D014357:Trypsin / Q000378:metabolism*
D009211:Myoglobin; D011506:Proteins; D014357:Trypsin