Implementation of targeted proteomics methods for the characterization of peptides and proteins in liquid biopsies

Abstract

Targeted proteomics methods emerged to detect and quantify well-defined sets of peptides with a high degree of specificity and sensitivity. Internal standard triggered-PRM (IS-PRM) was designed to increase the number of peptides monitored in one analysis without affecting the quality of the data obtained by using stable isotopically labeled (SIL) peptides as internal standards. We aimed to investigate the technical reproducibility of a type of IS-PRM method called SureQuant and to quantify the added SIL peptides and their corresponding endogenous forms. Three aliquots of commercially available human serum samples were digested with trypsin and a set of 802 isotopically-labelled standard (SIL) peptides were spiked-in to later be analyzed by LC-MS using SureQuant acquisition method. Instrument variability showed a coefficient of variation (CV) of 9.3%, whereas samplepreparation variability increases up to 34.7% and 39.1%. The technique enabled to detect, on average, 772 SIL peptides and 738 endogenous forms. Data obtained shows very good instrument reproducibility, whereas sample preparation procedure presents greater variability. Thus, this study proves the need for an improvement to achieve reproducibility and obtain reliable quantitative results.