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  • 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.
    Author: Jakoby T, Tholey A, van den Berg BH.
    Journal: Rapid Commun Mass Spectrom; 2012 Dec 15; 26(23):2777-85. PubMed ID: 23124669.
    Abstract:
    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.
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