The use of tryptic marker-peptides for the quantitative analysis of cystatin C.
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).
Storme
Michael L
ML
Laboratory of Medical Biochemistry and Clinical Analysis, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium. Michael.Storme@UGent.be
Sinnaeve
Bart A
BA
Van Bocxlaer
Jan F
JF
D000595:Amino Acid Sequence; D015415:Biomarkers / Q000032:analysis; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D055316:Cystatin C; D015891:Cystatins / Q000737:chemistry* / Q000302:isolation & purification; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000302:isolation & purification; D014357:Trypsin
D015415:Biomarkers; D055316:Cystatin C; D015891:Cystatins; D010446:Peptide Fragments; D014357:Trypsin