Matrix-assisted laser desorption/ionization-tandem mass spectrometry with high resolution and sensitivity for identification and characterization of proteins.
Although peptide mass fingerprinting is currently the method of choice to identify proteins, the number of proteins available in databases is increasing constantly, and hence, the advantage of having sequence data on a selected peptide, in order to increase the effectiveness of database searching, is more crucial. Until recently, the ability to identify proteins based on the peptide sequence was essentially limited to the use of electrospray ionization tandem mass spectrometry (MS) methods. The recent development of new instruments with matrix-assisted laser desorption/ionization (MALDI) sources and true tandem mass spectrometry (MS/MS) capabilities creates the capacity to obtain high quality tandem mass spectra of peptides. In this work, using the new high resolution tandem time of flight MALDI-(TOF/TOF) mass spectrometer from Applied Biosystems, examples of successful identification and characterization of bovine heart proteins (SWISS-PROT entries: P02192, Q9XSC6, P13620) separated by two-dimensional electrophoresis and blotted onto polyvinylidene difluoride membrane are described. Tryptic protein digests were analyzed by MALDI-TOF to identify peptide masses afterward used for MS/MS. Subsequent high energy MALDI-TOF/TOF collision-induced dissociation spectra were recorded on selected ions. All data, both MS and MS/MS, were recorded on the same instrument. Tandem mass spectra were submitted to database searching using MS-Tag or were manually de novo sequenced. An interesting modification of a tryptophan residue, a "double oxidation", came to light during these analyses.
Bienvenut
Willy V
WV
Central Clinical Chemistry Laboratory, Geneva University Hospital, Geneva, Switzerland. william.bienvenut@dim.hcuge.ch
Déon
Catherine
C
Pasquarello
Carla
C
Campbell
Jennifer M
JM
Sanchez
Jean-Charles
JC
Vestal
Marvin L
ML
Hochstrasser
Denis F
DF
D000818:Animals; D002417:Cattle; D030562:Databases, Protein; D015180:Electrophoresis, Gel, Two-Dimensional; D006352:Heart Ventricles / Q000737:chemistry; D015394:Molecular Structure; D010084:Oxidation-Reduction; D010449:Peptide Mapping / Q000295:instrumentation / Q000379:methods*; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000295:instrumentation / Q000379:methods; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism
D011506:Proteins; D014364:Tryptophan