title: Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents.
abstract: We describe here a multiplexed protein quantitation strategy that provides relative and absolute measurements of proteins in complex mixtures. At the core of this methodology is a multiplexed set of isobaric reagents that yield amine-derivatized peptides. The derivatized peptides are indistinguishable in MS, but exhibit intense low-mass MS/MS signature ions that support quantitation. In this study, we have examined the global protein expression of a wild-type yeast strain and the isogenic upf1Delta and xrn1Delta mutant strains that are defective in the nonsense-mediated mRNA decay and the general 5' to 3' decay pathways, respectively. We also demonstrate the use of 4-fold multiplexing to enable relative protein measurements simultaneously with determination of absolute levels of a target protein using synthetic isobaric peptide standards. We find that inactivation of Upf1p and Xrn1p causes common as well as unique effects on protein expression.
authors.affiliation: Applied Biosystems, Framingham, MA 01701, USA.
authors.forename: Philip L
authors.initials: PL
authors.lastname: Ross
authors.forename: Yulin N
authors.initials: YN
authors.lastname: Huang
authors.forename: Jason N
authors.initials: JN
authors.lastname: Marchese
authors.forename: Brian
authors.initials: B
authors.lastname: Williamson
authors.forename: Kenneth
authors.initials: K
authors.lastname: Parker
authors.forename: Stephen
authors.initials: S
authors.lastname: Hattan
authors.forename: Nikita
authors.initials: N
authors.lastname: Khainovski
authors.forename: Sasi
authors.initials: S
authors.lastname: Pillai
authors.forename: Subhakar
authors.initials: S
authors.lastname: Dey
authors.forename: Scott
authors.initials: S
authors.lastname: Daniels
authors.forename: Subhasish
authors.initials: S
authors.lastname: Purkayastha
authors.forename: Peter
authors.initials: P
authors.lastname: Juhasz
authors.forename: Stephen
authors.initials: S
authors.lastname: Martin
authors.forename: Michael
authors.initials: M
authors.lastname: Bartlet-Jones
authors.forename: Feng
authors.initials: F
authors.lastname: He
authors.forename: Allan
authors.initials: A
authors.lastname: Jacobson
authors.forename: Darryl J
authors.initials: DJ
authors.lastname: Pappin
mesh_terms: D002412:Cations; D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid; D015536:Down-Regulation; D005095:Exoribonucleases / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry; D007202:Indicators and Reagents / Q000494:pharmacology; D007477:Ions; D013058:Mass Spectrometry; D008956:Models, Chemical; D010455:Peptides / Q000737:chemistry; D010641:Phenotype; D040901:Proteomics / Q000379:methods*; D020365:RNA Helicases / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism; D013388:Succinimides / Q000737:chemistry
chemical_list: D002412:Cations; D005656:Fungal Proteins; D007202:Indicators and Reagents; D007477:Ions; D010455:Peptides; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; D013388:Succinimides; D005095:Exoribonucleases; C068398:XRN1 protein, S cerevisiae; C074151:NAM7 protein, S cerevisiae; D020365:RNA Helicases; C001426:N-hydroxysuccinimide