Protein identification via ion-trap collision-induced dissociation and examination of low-mass product ions.
A whole-protein tandem mass spectrometry approach for protein identification based on precursor ion charge state concentration via ion/ion reactions, ion-trap collisional activation, ion/ion proton-transfer reactions involving the product ions, and mass analysis over a narrow m/z range (up to m/z 2000) is described and evaluated. The experiments were carried out with a commercially available electrospray ion-trap instrument that has been modified to allow for ion/ion reactions. Reaction conditions and the approach to searching protein databases were developed with the assumption that the resolving power of the mass analyzer is insufficient to distinguish charge states on the basis of the isotope spacings. Ions derived from several charge states of cytochrome c, myoglobin, ribonuclease A, and ubiquitin were used to evaluate the approach for protein identification and to develop a two-step procedure to database searching to optimize specificity. The approach developed with the model proteins was then applied to whole cell lysate fractions of Saccharomyces cerevisiae. The results are illustrated with examples of assignments made for three a priori unknown proteins, each selected randomly from a lysate fraction. Two of the three proteins were assigned to species present in the database, whereas one did not match well any database entry. The combination of the mass measurement and the product ion masses suggested the possibility for the oxidation of two methionine residues of a protein in the database. The examples show that this limited whole-protein characterization approach can provide insights that might otherwise be lacking with approaches based on complete enzymatic digestion.
Bowers
Jeremiah J
JJ
Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, IN 47907-2084, USA.
Liu
Jian
J
Gunawardena
Harsha P
HP
McLuckey
Scott A
SA
D000818:Animals; D002417:Cattle; D045304:Cytochromes c / Q000737:chemistry; D016208:Databases, Factual; D009211:Myoglobin / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D040901:Proteomics; D012259:Ribonuclease, Pancreatic / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D025801:Ubiquitin / Q000737:chemistry
D009211:Myoglobin; D011506:Proteins; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D045304:Cytochromes c; D012259:Ribonuclease, Pancreatic