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Media type:
E-Article
Title:
Triplex protein quantification based on stable isotope labeling by peptide dimethylation applied to cell and tissue lysates
Contributor:
Boersema, Paul J.;
Aye, Thin Thin;
van Veen, Toon A. B.;
Heck, Albert J. R.;
Mohammed, Shabaz
Published:
Wiley, 2008
Published in:
PROTEOMICS, 8 (2008) 22, Seite 4624-4632
Language:
English
DOI:
10.1002/pmic.200800297
ISSN:
1615-9853;
1615-9861
Origination:
Footnote:
Description:
AbstractStable isotope labeling is at present one of the most powerful methods in quantitative proteomics. Stable isotope labeling has been performed at both the protein as well as the peptide level using either metabolic or chemical labeling. Here, we present a straightforward and cost‐effective triplex quantification method that is based on stable isotope dimethyl labeling at the peptide level. Herein, all proteolytic peptides are chemically labeled at their α‐ and ϵ‐amino groups. We use three different isotopomers of formaldehyde to enable the parallel analysis of three different samples. These labels provide a minimum of 4 Da mass difference between peaks in the generated peptide triplets. The method was evaluated based on the quantitative analysis of a cell lysate, using a typical “shotgun” proteomics experiment. While peptide complexity was increased by introducing three labels, still more than 1300 proteins could be identified using 60 μg of starting material, whereby more than 600 proteins could be quantified using at least four peptides per protein. The triplex labeling was further utilized to distinguish specific from aspecific cAMP binding proteins in a chemical proteomics experiment using immobilized cAMP. Thereby, differences in abundance ratio of more than two orders of magnitude could be quantified.