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 p...

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Bibliographic Details
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
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Summary: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).
ISSN:1615-9306
1615-9314
DOI:10.1002/jssc.200500127