✍ Scribed by Michael L. Storme; Bart A. Sinnaeve; Jan F. Van Bocxlaer
No coin nor oath required. For personal study only.
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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