Comparative study of label and label-free techniques using shotgun proteomics for relative protein quantification
2013 (English)In: Journal of chromatography. B, ISSN 1570-0232, E-ISSN 1873-376X, Vol. 928, 83-92 p.Article in journal (Refereed) Published
The analytical performance of three different strategies, iTRAQ (isobaric tag for relative and absolute quantitation), dimethyl labeling (DML) and label free (LF) for relative protein quantification using shotgun proteomics have been evaluated. The methods have been explored using samples containing (i) Bovine proteins in known ratios and (ii) Bovine proteins in known ratios spiked into E.Coli. The latter case mimics the actual conditions in a typical biological sample with a few differentially expressed proteins and a bulk of proteins with unchanged ratios. Additionally, the evaluation was performed on both Q-TOF and LTQ-FTICR mass spectrometers. LF LTQ-FTICR was found to have the highest proteome coverage (94 %) while the highest accuracy based on the artificially regulated proteins was found for DML LTQ-FTICR (54%). A good linearity (r2: 0.61-0.96) was shown for all methods within selected dynamic ranges. All methods were found to consistently underestimate bovine protein ratios when matrix proteins were added. However LF LTQ-FTICR was more tolerant towards a compression effect. A single peptide was demonstrated to be sufficient for a reliable quantification using iTRAQ. A ranking system utilizing several parameters important for quantitative proteomics demonstrated that the overall performance of the five different methods were; DML LTQ-FTICR > iTRAQ QTOF > LF LTQ-FTICR > DML Q-TOF > LF Q-TOF.
Place, publisher, year, edition, pages
2013. Vol. 928, 83-92 p.
Relative quantification, Proteomics, Mass spectrometry, Stable isotope labeling, Label free
Research subject Analytical Chemistry
IdentifiersURN: urn:nbn:se:uu:diva-180105DOI: 10.1016/j.jchromb.2013.03.027ISI: 000319236700011OAI: oai:DiVA.org:uu-180105DiVA: diva2:548154
De två (2) sista författarna delar sistaförfattarskapet.2012-08-292012-08-292013-07-01Bibliographically approved