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

• Published in: Journal of separation science 2005-09, Vol.28 (14), p.1759-1763
• Main Authors: Storme, Michael L., Sinnaeve, Bart A., Van Bocxlaer, Jan F.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Biomarkers - analysis; Calibration; Chromatography, High Pressure Liquid - methods; Cystatin C; Cystatins - chemistry; Cystatins - isolation & purification; Fundamental and applied biological sciences. Psychology; General aspects, investigation methods; Investigative techniques, diagnostic techniques (general aspects); LC-MS/MS; Marker-peptides; Mass Spectrometry - methods; Medical sciences; Miscellaneous. Technology; Molecular Sequence Data; Pathology. Cytology. Biochemistry. Spectrometry. Miscellaneous investigative techniques; Peptide Fragments - chemistry; Peptide Fragments - isolation & purification; Protein; Proteins; Quantitative analysis; Trypsin; Trypsinisation
• ISSN: 1615-9306; 1615-9314
• DOI: 10.1002/jssc.200500127

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).