title: Relative quantitation of proteins fractionated by the ProteomeLab PF 2D system using isobaric tags for relative and absolute quantitation (iTRAQ).
abstract: We describe an optimised protocol for application of isobaric tags for relative and absolute quantitation (iTRAQ) and tandem mass spectrometry to obtain relative quantitative data from peptides derived from tryptic digestions of proteins fractionated by using the 2D liquid-phase ProteomeLab PF 2D technique. This methodology is suitable for the quantitation of proteins from a pool of co-eluting proteins which are often difficult to identify for the purpose of candidate protein selection for biologically relevant qualitative/quantitative changes under experimental conditions or in disease states. iTRAQ quantitation also facilitates the possibility of result to result comparison using other methodologies such as UV protein quantitation via the ProteomeLab PF 2D technique. The optimised protocol outlined here allows relative quantitation by MALDI-TOF/TOF mass spectrometry with high sensitivity and without the need to perform 2D HPLC separation of labelled peptides. The overall outcome is the simplification in the data complexity and the ease of use of the labelling protocol.
authors.affiliation: Institute of Animal Physiology and Genetics, Academy of Sciences of Czech Republic, Rumburska 89, 277 21 Libechov, Czech Republic.
authors.forename: Helena
authors.initials: H
authors.lastname: Skalnikova
authors.forename: Pavel
authors.initials: P
authors.lastname: Rehulka
authors.forename: Josef
authors.initials: J
authors.lastname: Chmelik
authors.forename: Jirina
authors.initials: J
authors.lastname: Martinkova
authors.forename: Michaela
authors.initials: M
authors.lastname: Zilvarova
authors.forename: Suresh Jivan
authors.initials: SJ
authors.lastname: Gadher
authors.forename: Hana
authors.initials: H
authors.lastname: Kovarova
mesh_terms: D011506:Proteins / Q000032:analysis*; D011933:Reagent Kits, Diagnostic; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
chemical_list: D011506:Proteins; D011933:Reagent Kits, Diagnostic; D014357:Trypsin