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The use of tryptic marker-peptides for the quantitative analysis of cystatin C.

Authors
  • Storme, Michael L
  • Sinnaeve, Bart A
  • Van Bocxlaer, Jan F
Type
Published Article
Journal
Journal of separation science
Publication Date
Sep 01, 2005
Volume
28
Issue
14
Pages
1759–1763
Identifiers
PMID: 16224971
Source
Medline
License
Unknown

Abstract

The use of marker-peptides, measured by LC-MS/MS, is investigated for the quantitative analysis of proteins. To that end, cystatin C is chosen as a model protein. It not only functions as a proof of concept protein but the growing interest in cystatin C as a new marker of kidney failure provides a practical application at the same time. The use of trypsin-based proteolysis, to obtain so-called marker-peptides, simplifies the quantification of a protein to the quantification of a single or a number of peptides. Reproducibility of the trypsin proteolysis procedure is vital and has been optimised. A number of the marker-peptides obtained are selected for LC-MS(/MS) analysis. They are completely separated by high-pressure LC allowing maximum selectivity and mass spectrometric multiple reaction monitoring sensitivity. By doing so, linear calibration curves can be obtained for cystatin C over two orders of magnitude. Experiments have been performed on a triple quadrupole mass spectrometer by single ion monitoring (maximum sensitivity) as well as by multiple reaction monitoring (maximum specificity).

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