Identification and sequencing analysis of intact proteins via collision-induced dissociation and quadrupole time-of-flight mass spectrometry.
Identifying unknown proteins has become a central focal point for proteomic and biopharmaceutical development laboratories. Our laboratory investigated using quadrupole time-of-flight mass spectrometry (Qq/TOFMS) for the analysis of intact proteins for the purpose of identifying unknowns while limiting the number of sample-handling steps between protein extraction and identification. Eight standard proteins, both unmodified and disulfide-bonded and ranging in mass from 5 to 66 kDa, were analyzed using nanoelectrospray and collision-induced dissociation to generate peptide sequence tags. An MS analysis, followed by MS/MS analyses on two to five individual protein charge states, were obtained to make an identification. Peptide sequence tags were extracted from the MS/MS data and used, in conjunction with molecular mass and source origin, to obtain protein identifications using the web-based search engine ProteinInfo (www.proteometrics.com). All of the proteins were unambiguously identified from the input data, after which, all of the major product ions were identified for structural information. In most cases, N- and/or C-terminal ions, and also stretches of consecutive product ions from the protein interior, were observed. This method was applied to the analysis and identification of an unknown detected via reversed-phase high-performance liquid chromatography.
Nemeth-Cawley
Jennifer F
JF
Wyeth BioPharma, One Burtt Road, Andover, MA 01810, USA. jcawley@genetics.com
Rouse
Jason C
JC
D000595:Amino Acid Sequence; D016208:Databases, Factual; D004220:Disulfides / Q000032:analysis; D008401:Gas Chromatography-Mass Spectrometry; D007202:Indicators and Reagents; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011506:Proteins / Q000032:analysis*; D011994:Recombinant Proteins / Q000032:analysis; D017421:Sequence Analysis
D004220:Disulfides; D007202:Indicators and Reagents; D011506:Proteins; D011994:Recombinant Proteins