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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
Author(s) -
Soskic Vukic,
GodovacZimmermann Jasminka
Publication year - 2001
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/1615-9861(200111)1:11<1364::aid-prot1364>3.0.co;2-h
Subject(s) - chemistry , mass spectrometry , chromatography , bottom up proteomics , sample preparation in mass spectrometry , matrix assisted laser desorption/ionization , protein mass spectrometry , peptide , trypsin , peptide mass fingerprinting , capillary electrophoresis–mass spectrometry , gel electrophoresis , time of flight mass spectrometry , proteomics , desorption , tandem mass spectrometry , electrospray ionization , biochemistry , enzyme , ionization , organic chemistry , ion , adsorption , gene
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.