title: Improved reporter ion assignment of raw isobaric stable isotope labeled liquid chromatography/matrix-assisted laser desorption/ionization tandem time-of-flight mass spectral data for quantitative proteomics.
abstract: BACKGROUND
  Isobaric labeling strategies (e.g. iTRAQ or TMT) are commonly applied in tandem mass spectrometric (MS/MS) level quantitative proteomics. However, we frequently observed missing isotope reporter ion signals in a large-scale liquid chromatography/matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometric (LC/MALDI-TOF/TOF) quantitative proteomics experiment. To understand this issue, we systematically investigated the processing of MS/MS spectra into peak lists prior to peptide identification and quantification.
  
  
  METHODS
  A 15-protein standard, with six proteins in different concentrations, was labeled with iTRAQ 4-plex, iTRAQ 8-plex or TMT 6-plex, tryptic digested and measured using LC/MALDI-TOF/TOF. Three commercially and open-source available peak list generation software tools were compared based on missing reporter ions, peptide identification and quantification.
  
  
  RESULTS
  We found that each tool discarded lower-intensity reporter ions, when they followed a higher intensity reporter ion, due to the implemented de-isotoping algorithms. By using the non-de-isotoping setting within TS2Mascot, we found that all reporter ions are exported, yet less peptides were identified with Mascot. Therefore, we developed a strategy merging the de-isotoped and non-de-isotoped outputs from TS2Mascot using the Perl script RICmerge.pl.
  
  
  CONCLUSIONS
  With this approach, we correctly quantified all labeled peptides that were identified within the 15-protein standard. This strategy allows improved annotation of isobaric tag labeled peptide MS/MS spectra and improves downstream peptide and protein quantification in proteomics studies.
authors.affiliation: AG Systematische Proteomforschung & Bioanalytik, Institut für Experimentelle Medizin, Christian-Albrechts-Universität zu Kiel, Kiel, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Jakoby
authors.forename: Andreas
authors.initials: A
authors.lastname: Tholey
authors.forename: Bart H J
authors.initials: BH
authors.lastname: van den Berg
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006801:Humans; D010455:Peptides / Q000032:analysis; D011506:Proteins / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D010455:Peptides; D011506:Proteins