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Effect of experimental conditions on the analysis of sodium dodecyl sulfate polyacrylamide gel electrophoresis separated proteins by matrix-assisted laser desorption/ionization mass spectrometry



Effect of experimental conditions on the analysis of sodium dodecyl sulfate polyacrylamide gel electrophoresis separated proteins by matrix-assisted laser desorption/ionization mass spectrometry



Rapid Communications in Mass Spectrometry 14(1): 18-25



Two mixtures of proteins having molecular weights in the range of ~8 to 97 kDa (the low molecular weight mixture comprising ubiquitin, alpha -lactalbumin, myoglobin, beta -lactoglobulin B, trypsinogen, and carbonic anhydrase (carbonate dehydratase), and the high molecular weight mixture comprising phosphorylase b, transferrin, serum albumin, and peroxidase) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and examined by delayed extraction matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Part of the aim in this study is to gain more insight into the influence of the various experimental conditions on the overall quality of the acquired mass spectral data. Different protein extraction procedures, 2 staining agents, and extraction times, were among the parameters assessed. In terms of the overall quality of the acquired mass spectra and the speed of protein recovery, ultrasonic assisted passive elution, into a solvent mixture containing formic acid/acetonitrile/2-isopropanol/water, was found to be more efficient than other elution procedures. The higher resolution associated with the delayed extraction mode allowed the identification of a number of protein modifications, including multiple formylation provoked by formic acid, cysteine alkylation caused by unpolymerized acrylamide monomers, and complexation with the staining reagents. The detection of these modifications, however, was limited to proteins under 30 kDa. Analysis of a ubiquitin tryptic digest by reflectron MALDI time-of-flight (TOF) allowed reliable identification of a number of formylation sites.

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Accession: 003727991

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DOI: 10.1002/(SICI)1097-0231(20000115)14:1<18::AID-RCM826>3.3.CO;2-E



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