title: Comparison of 4-plex to 8-plex iTRAQ quantitative measurements of proteins in human plasma samples.
abstract: Methods for isobaric tagging of peptides, iTRAQ or TMT, are commonly used platforms in mass spectrometry based quantitative proteomics. These two methods are very often used to quantitate proteins in complex samples, e.g., serum/plasma or CSF supporting biomarker discovery studies. The success of these studies depends on multiple factors, including the accuracy of ratios of reporter ions reflecting quantitative changes of proteins. Because reporter ions are generated during peptide fragmentation, the differences of chemical structure of iTRAQ balance groups may have an effect on how efficiently these groups are fragmented and thus how differences in protein expression will be measured. Because 4-plex and 8-plex iTRAQ reagents do have different structures of balanced groups, it has been postulated that indeed differences in protein identification and quantitation exist between these two reagents. In this study we controlled the ratios of tagged samples and compared quantitation of proteins using 4-plex versus 8-plex reagents in the context of a highly complex sample of human plasma using ABSciex 4800 MALDI-TOF/TOF mass spectrometer and ProteinPilot 4.0 software. We observed that 8-plex tagging provides more consistent ratios than 4-plex without compromising protein identification, thus allowing investigation of eight experimental conditions in one analytical experiment.
authors.affiliation: Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, Nebraska 68198-5800, USA.
authors.forename: Gwenael
authors.initials: G
authors.lastname: Pottiez
authors.forename: Jayme
authors.initials: J
authors.lastname: Wiederin
authors.forename: Howard S
authors.initials: HS
authors.lastname: Fox
authors.forename: Pawel
authors.initials: P
authors.lastname: Ciborowski
mesh_terms: D001798:Blood Proteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D010446:Peptide Fragments / Q000737:chemistry; D059748:Proteolysis; D020543:Proteome / Q000737:chemistry* / Q000378:metabolism; D040901:Proteomics; D013194:Staining and Labeling; D053719:Tandem Mass Spectrometry
chemical_list: D001798:Blood Proteins; D010446:Peptide Fragments; D020543:Proteome