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Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS

Title data

Gerber, Scott A. ; Rusch, John ; Stemmann, Olaf ; Kirschner, Marc W. ; Gygi, Steven P.:
Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS.
In: Proceedings of the National Academy of Sciences of the United States of America. Vol. 100 (10 June 2003) Issue 12 . - pp. 6940-6945.
ISSN 1091-6490
DOI: https://doi.org/10.1073/pnas.0832254100

Official URL: Volltext

Abstract in another language

A need exists for technologies that permit the direct quantification of differences in protein and posttranslationally modified protein expression levels. Here we present a strategy for the absolute quantification (termed AQUA) of proteins and their modification states. Peptides are synthesized with incorporated stable isotopes as ideal internal standards to mimic native peptides formed by proteolysis. These synthetic peptides can also be prepared with covalent modifications (e.g., phosphorylation, methylation, acetylation, etc.) that are chemically identical to naturally occurring posttranslational modifications. Such AQUA internal standard peptides are then used to precisely and quantitatively measure the absolute levels of proteins and posttranslationally modified proteins after proteolysis by using a selected reaction monitoring analysis in a tandem mass spectrometer. In the present work, the AQUA strategy was used to (i) quantify low abundance yeast proteins involved in gene silencing, (ii) quantitatively determine the cell cycle-dependent phosphorylation of Ser-1126 of human separase protein, and (iii) identify kinases capable of phosphorylating Ser-1501 of separase in an in vitro kinase assay. The methods described here represent focused, alternative approaches for studying the dynamically changing proteome.

Further data

Item Type: Article in a journal
Refereed: Yes
Institutions of the University: Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Biology > Chair Genetics > Chair Genetics - Univ.-Prof. Dr. Olaf Stemmann
Profile Fields > Advanced Fields > Molecular Biosciences
Research Institutions > Research Centres > Bayreuth Center for Molecular Biosciences - BZMB
Faculties
Faculties > Faculty of Biology, Chemistry and Earth Sciences
Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Biology
Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Biology > Chair Genetics
Profile Fields
Profile Fields > Advanced Fields
Research Institutions
Research Institutions > Research Centres
Result of work at the UBT: No
DDC Subjects: 500 Science > 570 Life sciences, biology
Date Deposited: 27 Mar 2015 08:06
Last Modified: 27 Mar 2015 08:06
URI: https://eref.uni-bayreuth.de/id/eprint/8557