The use of tryptic marker-peptides for the quantitative analysis of cystatin C |
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Authors: | Storme Michael L Sinnaeve Bart A Van Bocxlaer Jan F |
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Affiliation: | Laboratory of Medical Biochemistry and Clinical Analysis, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium. Michael.Storme@UGent.be |
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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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