Deep plasma proteomics profiling of healthy humans at two different stages of adulthood

Mass spectrometry-based proteomics is a valuable strategy for plasma proteome profiling that has successfully been used in many personalized medicine and health monitoring studies. Despite its potential, analyzing plasma samples by mass spectrometry poses some analytical challenges, particularly due to the broad dynamic range of plasma, which limits access to its complete proteome. To overcome these limitations and enhance in-depth protein identification, several commercial solutions have been developed to increase the number of identified proteins and the portion of the accessible plasma proteome. However, beyond merely increasing the number of identified peptides and proteins, it is also crucial to demonstrate that these new approaches maintain the fidelity of protein relative quantification, thereby reproducing and extending previous biological insights obtained with neat plasma.

In this study, we compared the relative quantitation and biological information retrieved from neat and enriched human plasma in a cohort of healthy individuals at two different stages of adulthood. Plasma samples from 60 healthy individuals (30 aged 18-25, 30 aged 70-84) were analyzed by mass spectrometry using a data-independent acquisition (DIA) workflow. The study utilized neat and enriched plasma from the same individuals, processed with either the iST-BCT or ENRICH-iST PreOmics kits, and analyzed using a 60-minute gradient on an Orbitrap Eclipse instrument. Acquired data were analyzed using a library-free approach with DIA-NN 1.8.1 software.

Enrichment procedures resulted in more than a threefold increase in protein identification compared to neat plasma, with most proteins identified in neat plasma also detected in enriched samples. Moreover, our results show that the enrichment process does not increase quantitative variability and can reproduce most protein fold-changes previously observed. Finally, this approach provides deeper insights into age-related differences in the plasma proteome, adding valuable information from enriched samples.