Evaluation of endogenous plasma peptide extraction methods for mass spectrometric biomarker discovery.

Peptides have a role in the inflammatory response, tumor biology, and endocrine processes, presenting them as appealing biomarker candidates. However, peptide extraction efficacy for clinical profiling remains a pivotal technological challenge, as maximum coverage of the plasma peptidome is limited by a range of factors including the inherent complexity of human plasma and the lower concentration of peptides compared to abundant proteins. The aim of this study was to evaluate commonly employed peptide extraction methodologies in terms of total number of peptides detected and the mass range of peptides observed by MALDI. Despite showing coelution of proteins, solid-phase extraction (SPE) methods exhibited superior plasma peptide recovery than ultrafiltration, acetonitrile (ACN) precipitation, or size-exclusion chromatography methods under conditions employed in the study. Not surprisingly, in line with studies challenging the veracity of many peptide biomarker studies, the majority of identified peptides eluted from SPE methods corresponded to proteolytic truncations of the most abundant plasma proteins. The prefractionation of plasma with acetonitrile precipitation prior to SPE provided distinct ion signal profiles and is worthy of further study. In conclusion, this study favors the use of SPE in peptide extraction protocols for increased biomarker coverage and diversity from the plasma peptidome.