Identification of proteins in polyacrylamide gels by mass spectrometric peptide mapping combined with database search.
Mass spectrometric peptide mapping of proteins separated by one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis has been investigated. The best results are obtained after blotting of the proteins onto polyvinylidene difluoride membranes followed by enzymatic digestion of the protein on the membrane. The peptide maps were investigated in terms of completeness and applicability for protein identification using a previously developed database search program as well as for the possibility for full characterization of covalent modifications in the proteins. The most complete peptide maps were obtained when the proteins were reduced and alkylated on the membrane prior to enzymatic digestion followed by separation of the resulting mixture by high performance liquid chromatography prior to mass spectrometric analysis. Such peptide maps cover up to 98% of the sequence and consequently may allow complete characterization of post-translational modifications in proteins for which the amino acid sequence is known. The fastest and most sensitive procedure to obtain peptide maps sufficient for protein identification was direct analysis of the extracted peptide mixture by matrix-assisted laser desorption ionization (MALDI) mass spectrometry. The use of external and internal calibration of MALDI spectra for database searches is evaluated as well as the possibility of including a post-calibration routine within the search program.
Mørtz
E
E
Department of Molecular Biology, Odense University, Denmark.
Vorm
O
O
Mann
M
M
Roepstorff
P
P
D000478:Alkylation; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D016208:Databases, Factual; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D007834:Lasers; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D011506:Proteins / Q000032:analysis*; D014357:Trypsin
D011506:Proteins; D014357:Trypsin