Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents.
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.
Ross
Philip L
PL
Applied Biosystems, Framingham, MA 01701, USA.
Huang
Yulin N
YN
Marchese
Jason N
JN
Williamson
Brian
B
Parker
Kenneth
K
Hattan
Stephen
S
Khainovski
Nikita
N
Pillai
Sasi
S
Dey
Subhakar
S
Daniels
Scott
S
Purkayastha
Subhasish
S
Juhasz
Peter
P
Martin
Stephen
S
Bartlet-Jones
Michael
M
He
Feng
F
Jacobson
Allan
A
Pappin
Darryl J
DJ
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
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