Effect of experimental conditions on the analysis of sodium dodecyl sulphate polyacrylamide gel electrophoresis separated proteins by matrix-assisted laser desorption/ ionisation mass spectrometry.
Two mixtures of proteins having molecular weights in the range approximately 8-97 kDa were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and examined by delayed extraction matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS). Part of our 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, two 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 unpolymerised 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 the formylation sites.
Galvani
M
M
GlaxoWellcome Medicines Research Centre, via Fleming 4, Verona, 37135 Italy.
Bordini
E
E
Piubelli
C
C
Hamdan
M
M
D000818:Animals; D002256:Carbonic Anhydrases / Q000032:analysis / Q000737:chemistry; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D007768:Lactalbumin / Q000032:analysis / Q000737:chemistry; D007782:Lactoglobulins / Q000032:analysis / Q000737:chemistry; D008970:Molecular Weight; D009211:Myoglobin / Q000032:analysis / Q000737:chemistry; D012967:Sodium Dodecyl Sulfate; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013501:Surface-Active Agents; D014362:Trypsinogen / Q000032:analysis / Q000737:chemistry; D014452:Ubiquitins / Q000032:analysis* / Q000737:chemistry
D007782:Lactoglobulins; D009211:Myoglobin; D013501:Surface-Active Agents; D014452:Ubiquitins; D012967:Sodium Dodecyl Sulfate; D014362:Trypsinogen; D007768:Lactalbumin; D002256:Carbonic Anhydrases