Absolute Quantitative Targeted Proteomics Assays for Plasma Proteins

Preclinical and clinical trials require rapid, precise, and multiplexed analytical methods to characterize the complex samples and to allow high-throughput biomarker monitoring with low consumption of sample material. Targeted proteomics has been used to address these challenges when quantifying protein abundances in complex biological matrices. In many of these studies, blood plasma is collected either as the main research or diagnostic sample or in combination with other specimens. Mass spectrometry (MS)-based targeted proteomics using multiple reaction monitoring (MRM) or parallel reaction monitoring (PRM) with stable isotope-labeled internal standard (SIS) peptides allows robust characterization of blood plasma protein via absolute quantification. Compared to other commonly used technologies like enzyme-linked immunosorbent assay (ELISA), targeted proteomics is faster, more sensitive, and more cost-effective. Here we describe a protocol for the quantification of proteins in blood plasma using targeted MRM proteomics with heavy-labeled internal standards. The 270-protein panel allows rapid and robust absolute quantitative proteomic characterization of blood plasma in a 1 h gradient. The method we describe here works for non-depleted plasma, which makes it simple and easy to implement. Moreover, the protocol works with the two most commonly used blood plasma collection methods used in practice, namely, either K2EDTA or sodium citrate as anticoagulants.