Matrix-assisted laser desorption/ionization mass spectrometry of proteins extracted directly from sodium dodecyl sulphate-polyacrylamide gels.
A new strategy for the characterization of Coomassie Brilliant Blue stained SDS-PAGE separated proteins by UV-MALDI-MS is reported. The proteins are extracted directly from the polyacrylamide gel by treatment with an organic solvent mixture consisting of formic acid, acetonitrile, isopropanol and water in an ultrasonic bath. A fraction of the supernatant is then mixed directly with the matrix solution and measured by MALDI-MS. High quality spectra could be obtained from gels which were loaded with 6 pmol of myoglobin. Compared to other methods based on electroblotting or electroelution this method is much simpler and less time consuming. The sensitivity is higher than or comparable to the Coomassie Blue staining procedure for proteins up to about 25 kDa. Another advantage is that mass shifts due to charging effects of the membranes, which are common if membranes are mounted directly on the sample target, can be avoided. However, all proteins studied showed slightly higher masses than expected which reduces mass accuracy to 0.2-0.3%. This is presumably partly due to formylation of serine or threonine residues during incubation in formic acid. Gel electrophoresis induced modifications can contribute as well. The possibility of further characterizing the remaining part of the supernatant after extraction by means of proteolytic digestion is also demonstrated. The knowledge of both molecular weight of the whole protein and of the proteolytic fragments increases specificity for protein identification by searching in sequence databases.
Ehring
H
H
Pharmacia & Upjohn, Department of Structural Chemistry, Stockholm, Sweden.
Strömberg
S
S
Tjernberg
A
A
Norén
B
B
D002256:Carbonic Anhydrases / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D007202:Indicators and Reagents; D009211:Myoglobin / Q000737:chemistry; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification; D012394:Rosaniline Dyes; D012967:Sodium Dodecyl Sulfate; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013056:Spectrophotometry, Ultraviolet; D014452:Ubiquitins / Q000737:chemistry
D007202:Indicators and Reagents; D009211:Myoglobin; D011506:Proteins; D012394:Rosaniline Dyes; D014452:Ubiquitins; D012967:Sodium Dodecyl Sulfate; D002256:Carbonic Anhydrases; C004692:coomassie Brilliant Blue