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Novel and cost‐effective 6‐plex isobaric tagging reagent, DiART, is effective for identification and relative quantification of complex protein mixtures using PQD fragmentation
Author(s) -
Ramsubramaniam Nikhil,
Tao Feng,
Li Shuwei,
Marten Mark R.
Publication year - 2013
Publication title -
journal of mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.475
H-Index - 121
eISSN - 1096-9888
pISSN - 1076-5174
DOI - 10.1002/jms.3249
Subject(s) - chemistry , reagent , isobaric labeling , quantitative proteomics , chromatography , mass spectrometry , tandem mass tag , isobaric process , tandem mass spectrometry , quadrupole ion trap , ion trap , stable isotope labeling by amino acids in cell culture , fragmentation (computing) , analytical chemistry (journal) , protein mass spectrometry , proteomics , biochemistry , organic chemistry , physics , gene , thermodynamics , operating system , computer science
Deuterium isobaric Amine Reactive Tag (DiART) reagents facilitate relative quantification during proteomic analysis in a functionally similar manner to commercially available isobaric tag for relative and absolute quantitation (iTRAQ) and tandem mass tag (TMT) reagents. In contrast to iTRAQ and TMT, DiART reagents incorporate deuterium isotopes which significantly reduce the number of required synthesis steps and hence have potential to significantly reduce reagent production cost. We examined the capability of DiART for performing quantitative proteomic experiments using a linear ion‐trap mass spectrometer with Pulsed Q Dissociation (PQD) fragmentation. Using a synthetic peptide tagged with DiART reagent, we observed a precise mass shift of 144.79 Da on the triply charged precursor ion, which shows complete derivatization of the N‐terminus and ε‐amino group of lysine. A DiART tagged tryptic digest of bovine serum albumin showed a sequence coverage of 57.99% which was very comparable to that showed by iTRAQ, 54.77%. Furthermore, a ten protein mixture tagged with DiART reagents and mixed in 1:1:1:1:1:1 exhibited < 15% error, whereas a linear trend (slope of 1.085) was observed when tagged proteins were mixed in the ratio 2:1:2:4:10:14 and plotted against theoretical ratios. Finally, when complex cell‐wall protein mixtures from the model fungus A. nidulans were tagged with DiART reagents and mixed in different ratios, they exhibited similar trends. We conclude that DiART reagents are capable of performing quantitative proteomic experiments using PQD on a linear ion trap mass spectrometer. Copyright © 2013 John Wiley & Sons, Ltd.