title: Comparison of protein expression ratios observed by sixplex and duplex TMT labeling method.
abstract: Stable isotope labeling via isobaric derivatization of peptides is a universally applicable approach that enables concurrent identification and quantification of proteins in different samples using tandem mass spectrometry. In this study, we evaluated the performance of amine-reactive isobaric tandem mass tag (TMT), available as duplex and sixplex sets, with regard to their ability to elucidate protein expression changes. Using rat brain tissue from two different developmental time points, postnatal day 1 (p1) and 45 (p45), as a model system, we compared the protein expression ratios (p45/p1) observed using duplex TMT tags in triplicate measurements versus sixplex tag in a single LC-MS/MS analysis. A correlation of 0.79 in relative protein abundance was observed in the proteins quantified by these two sets of reagents. However, more proteins passed the criteria for significant fold change (-1.0 ≤ log(2) ratio (p45/p1) ≥ +1.0 and p < 0.05) in the sixplex analysis. Nevertheless, in both methods most proteins showing significant fold change were identified by multiple spectra, increasing their quantification precision. Additionally, the fold change in p45 rats against p1, observed in TMT experiments, was corroborated by a metabolic labeling strategy where relative quantification of differentially expressed proteins was obtained using (15)N-labeled p45 rats as an internal standard.
authors.affiliation: Department of Chemical Physiology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
authors.forename: Navin
authors.initials: N
authors.lastname: Rauniyar
authors.forename: Benbo
authors.initials: B
authors.lastname: Gao
authors.forename: Daniel B
authors.initials: DB
authors.lastname: McClatchy
authors.forename: John R
authors.initials: JR
authors.lastname: Yates
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D001923:Brain Chemistry; D002853:Chromatography, Liquid; D018507:Gene Expression Regulation, Developmental; D007553:Isotope Labeling; D009419:Nerve Tissue Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D010446:Peptide Fragments / Q000032:analysis* / Q000737:chemistry; D040901:Proteomics; D051381:Rats; D017207:Rats, Sprague-Dawley; D013194:Staining and Labeling / Q000379:methods*; D053719:Tandem Mass Spectrometry
chemical_list: D009419:Nerve Tissue Proteins; D010446:Peptide Fragments