Improvement of an in-gel tryptic digestion method for matrix-assisted laser desorption/ionization-time of flight mass spectrometry peptide mapping by use of volatile solubilizing agents.
The combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), in-gel enzymatic digestion of proteins separated by two-dimensional gel electrophoresis and searches of molecular weight in peptide-mass databases is a powerful and well established method for protein identification in proteomics analysis. For successful protein identification by MALDI-TOF mass spectrometry of peptide mixtures, critical parameters include highly specific enzymatic cleavage, high mass accuracy and sufficient numbers and sequence coverage of the peptides which can be analyzed. For in-gel digestion with trypsin, the method employed should be compatible both with enzymatic cleavage and subsequent MALDI-TOF MS analysis. We report here an improved method for preparation of peptides for MALDI-TOF MS mass fingerprinting by using volatile solubilizing agents during the in-gel digestion procedure. Our study clearly demonstrates that modification of the in-gel digestion protocols by addition of dimethyl formamide (DMF) or a mixture of DMF/N,N-dimethyl acetamide at various concentrations can significantly increase the recovery of peptides. These higher yields of peptides resulted in more effective protein identification.
Soskic
V
V
Center for Molecular Medicine, Department of Medicine, University College London, UK.
Godovac-Zimmermann
J
J
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D004126:Dimethylformamide / Q000494:pharmacology; D008969:Molecular Sequence Data; D010449:Peptide Mapping / Q000379:methods*; D010455:Peptides / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D012709:Serum Albumin / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*
D010455:Peptides; D012709:Serum Albumin; D004126:Dimethylformamide; D014357:Trypsin