Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents

Ross, P. L., Huang, Y. N., Marchese, J. N., Williamson, B., Parker, K., Hattan, S., Khainovski, N., Pillai, S., Dey, S., Daniels, S., Purkayastha, S., Juhasz, P., Martin, S., Bartlet-Jones, M., He, F., Jacobson, A., Pappin, D. J. (December 2004) Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol Cell Proteomics, 3 (12). pp. 1154-69. ISSN 1535-9476 (Print)1535-9476 (Linking)

URL: http://www.ncbi.nlm.nih.gov/pubmed/15385600
DOI: 10.1074/mcp.M400129-MCP200

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.

Item Type: Paper
Uncontrolled Keywords: Cations Chromatography, Ion Exchange Chromatography, Liquid Down-Regulation Exoribonucleases/metabolism Fungal Proteins/chemistry Indicators and Reagents/pharmacology Ions Mass Spectrometry Models, Chemical Peptides/chemistry Phenotype Proteomics/ methods RNA Helicases/metabolism RNA, Messenger/metabolism Saccharomyces cerevisiae/ metabolism Saccharomyces cerevisiae Proteins/ chemistry/metabolism Succinimides/chemistry
Subjects: Investigative techniques and equipment
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification
organism description > bacteria
Investigative techniques and equipment > spectroscopy > mass spectrometry
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein characterization
Investigative techniques and equipment > spectroscopy
CSHL Authors:
Communities: CSHL labs > Pappin lab
Depositing User: Matt Covey
Date: December 2004
Date Deposited: 04 Mar 2013 20:54
Last Modified: 04 Mar 2013 20:54
Related URLs:
URI: http://repository.cshl.edu/id/eprint/27713

Actions (login required)

Administrator's edit/view item Administrator's edit/view item
CSHL HomeAbout CSHLResearchEducationNews & FeaturesCampus & Public EventsCareersGiving