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The use of tryptic marker‐peptides for the quantitative analysis of Cystatin C
Author(s) -
Storme Michael L.,
Sinnaeve Bart A.,
Van Bocxlaer Jan F.
Publication year - 2005
Publication title -
journal of separation science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.72
H-Index - 102
eISSN - 1615-9314
pISSN - 1615-9306
DOI - 10.1002/jssc.200500127
Subject(s) - chemistry , cystatin c , chromatography , mass spectrometry , triple quadrupole mass spectrometer , reproducibility , quantitative analysis (chemistry) , cystatin , selected reaction monitoring , trypsin , quantitative proteomics , proteolysis , peptide , analytical chemistry (journal) , tandem mass spectrometry , proteomics , biochemistry , enzyme , creatinine , gene
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).