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Simplifying sample handling for protein identification by peptide mass fingerprint using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry
Author(s) -
Cordeiro Francisco M.,
Carreira Ricardo J.,
RialOtero Raquel,
Rivas M. Gabriela,
Moura Isabel,
Capelo JoseLuis
Publication year - 2007
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.3214
Subject(s) - chemistry , mass spectrometry , chromatography , matrix assisted laser desorption/ionization , peptide mass fingerprinting , time of flight mass spectrometry , sample preparation in mass spectrometry , sample preparation , peptide , matrix (chemical analysis) , myoglobin , surface enhanced laser desorption/ionization , whey protein , desorption , analytical chemistry (journal) , ionization , biochemistry , proteomics , electrospray ionization , organic chemistry , ion , adsorption , gene
Abstract An ultrasonic bath, an ultrasonic probe and a sonoreactor were used to speed up the kinetics of the reactions involved in each step of the sample handling for in‐gel protein identification by peptide mass fingerprint, PMF, using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF‐MS). The following steps were successfully accelerated using ultrasonic energy: gel washing, protein reduction, and protein alkylation. As a result, a reduction comprising 80% to 90% of the total time involved in the classic approach was achieved. In addition the sample handling was also drastically simplified. The number of peptides identified and the protein sequence coverage obtained for the new procedure were comparable to those obtained with the traditional sample treatment for the following protein standards: glycogen phosphorylase b, BSA, ovalbumin, carbonic anhydrase, trypsin inhibitor and α ‐lactalbumin. Finally, as a proof of the procedure, specific proteins were identified from complex protein mixtures obtained from three different sulphate‐reducing bacteria: Desulfovibrio desulfuricans G20, Desulfuvibrio gigas NCIB 9332, and Desulfuvibrio desulfuricans ATCC 27774. Copyright © 2007 John Wiley & Sons, Ltd.