title: A new calibrant for matrix-assisted laser desorption/ionization time-of-flight-time-of-flight post-source decay tandem mass spectrometry of non-digested proteins for top-down proteomic analysis.
abstract: BACKGROUND
  Matrix-assisted laser desorption/ionization (MALDI) time-of-flight-time-of-flight (TOF-TOF) post-source decay (PSD) tandem mass spectrometry (MS/MS) has seen increasing use for analysis of non-digested protein ions for top-down proteomic identification. However, there is no commonly accepted calibrant for this purpose beyond the use of peptide calibrants whose fragment ions span a lower mass-to-charge (m/z) range.
  
  
  METHODS
  We have used the PSD-generated fragment ions of disulfide-reduced/alkylated thioredoxin (AlkTrx) for TOF-TOF calibration in reflectron mode for the purpose of PSD-MS/MS analysis. The average m/z values of AlkTrx fragment ions were used for calibration. The quality of the calibration was assessed from the observed fragment ion mass error of MS/MS of the YahO protein from an unfractionated bacterial cell lysate of Escherichia coli O157:H7 as well as from MS/MS of bovine ubiquitin. The fragment ion mass errors of these two analytes were also used to assess instrument calibration using the monoisotopic fragment ions of [Glu(1)]-fibrinopeptide B (GluFib).
  
  
  RESULTS
  A general improvement in fragment ion mass accuracy was observed using the AlkTrx calibration compared to the GluFib calibration which resulted in a more significant top-down proteomic identification of these analyte proteins.
  
  
  CONCLUSIONS
  Our results suggest that AlkTrx may be useful as a calibrant for MALDI-TOF-TOF-PSD-MS/MS of small and modest-sized protein ions. The uniform fragmentation efficiency of YahO across its sequence suggests that it may be useful as a post-calibration standard to assess PSD-MS/MS instrument performance as well as establishing appropriate top-down proteomic fragment ion tolerances.
authors.affiliation: Western Regional Research Center, Agricultural Research Service, US Department of Agriculture, Albany, CA 94710, USA. clifton.fagerquist@ars.usda.gov
authors.forename: Clifton K
authors.initials: CK
authors.lastname: Fagerquist
authors.forename: Omar
authors.initials: O
authors.lastname: Sultan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002138:Calibration; D002417:Cattle; D003198:Computer Simulation; D029968:Escherichia coli Proteins / Q000032:analysis / Q000737:chemistry; D008969:Molecular Sequence Data; D040901:Proteomics / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D025801:Ubiquitin / Q000032:analysis / Q000737:chemistry
chemical_list: D029968:Escherichia coli Proteins; D025801:Ubiquitin