Premium
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
Author(s) -
Jakoby Thomas,
Tholey Andreas,
Berg Bart H.J.
Publication year - 2012
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.6403
Subject(s) - chemistry , isobaric labeling , mass spectrometry , quantitative proteomics , chromatography , tandem mass spectrometry , tandem mass tag , proteomics , matrix assisted laser desorption/ionization , peptide , analytical chemistry (journal) , protein mass spectrometry , desorption , biochemistry , organic chemistry , adsorption , gene
RATIONALE 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. Copyright © 2012 John Wiley & Sons, Ltd.