An Improved Assay for Quantitation of Cerebrospinal Fluid Cystatin C Using Liquid Chromatography Tandem Mass...

Cystatin C (CST3) is expressed ubiquitously and implicated in several neurological diseases. It can be posttranscriptionally modified. CST3 is usually quantified in a biological sample using antibody-based methods. Posttranscriptional modification can hamper antibody-based detection systems by altering antibody-binding epitope(s). To circumvent this problem, enzymatic digestion and liquid chromatography tandem mass spectrometry (LC-MS/MS) technique can be employed to identify and measure peptides of a target protein in a complex biological mixture. This chapter describes an LC-MS/MS-based method for accurate measurement of CST3 in cerebrospinal fluid (CSF). Here, CSF was directly subjected to trypsin digestion and digested peptides were extracted using a solid-phase extraction column. Extracted peptide samples were directly used for LC-MS/MS-based identification and quantification of CST3 peptides. Comparing the concentration in a set of samples measured by LC-MS/MS with that of immunoassay shows that it was significantly higher when measured by LC-MS/MS method, suggesting it a better quantification method. This approach is particularly well suited when posttranscriptional modification of CST3 is suspected and sample volume of CSF is small.