Analysis of peptides in biological fluids by LC-MS/MS.

Abstract

Urine contains large amounts of small peptides, which may represent a rich, yet largely unexplored, source of novel biomarkers for disease monitoring. This chapter describes detailed procedures for the analysis of urinary polypeptides by LC-MS/MS. Hundreds to thousands of small peptides (∼700 to ∼7000 Da) can be detected in urine with the described techniques. Extraction procedures, based on commercially available reagents, effectively remove interfering urinary organic and inorganic salts and neutral compounds, making this a robust and simple assay with the power to detect hundreds to thousands of polypeptides in urine. Analysis time is relatively short, making this protocol a valuable alternative to conventional proteomic techniques based on multidimensional separations. The methodology is therefore particularly useful when the aim is to analyse samples with sufficient depth and throughput so as to make it useful to compare large numbers of specimens. Procedures for enhancing quantitative and qualitative analysis of LC-MS/MS data are also detailed.