Assistant: Apr. M. STORME

Quantitative analysis of proteins using liquid chromatography mass spectrometry and marker peptides

Nowadays, most proteomics applications are purely qualitative or semi-quantitative, e.g. using ICAT-technologies. However, there is a growing need for absolute quantitation of proteins, in particular due to the growing amount of so-called 'protein-medicins'. Also, the discovery of new protein-biomarkers increasingly demands quantitation. Nearly all diagnostic important proteins are now currently measured by immunoassays. Nevertheless, a certain lack of specificity is an important drawback. The use of mass spectrometry coupled to liquid chromatography could solve some of these problems.
In a new approach we seek to optimize an alternative technique to quantify proteins with LC-MS. To this end, we enzymatically cleave a given protein, typically with trypsin, and choose a unique set of marker peptides. These peptides, representing the protein of interest, are then quantified using LC/MS-MS with an internal standard. Our protein of interst is Cystatin C, an 146 aa protein of 13 kDa with two disulfid bridges between aa 99-104 and aa 123-143. Cystatin C is choosen as a model but also for his potential as a new marker of kidney failure.
Currently, we aim to develop a reproducible in solution tryptic digest procedure, as a first vital step in our marker peptide approach.

By Apr. Michael Storme