Impact of Variations in Commercial Standard Operating Procedure on Plasma Proteome Recovery

Advancements in mass spectrometry and complementary technologies
now enable comprehensive, high-resolution plasma proteomics. Plasma
is a key biofluid for clinical research, harboring potential disease-informative
biomarkers. Commercial sources of nondiseased, healthy donor plasma
samples are often used for proteomic workflow development and as controls
for clinical studies. The overarching assumption is that standard
operating procedures for plasma preparation are comparable across
different sources. In this study, we investigated the effectiveness
of a particle-based protein enrichment strategy against a conventional
proteomics workflow on plasma samples from five commonly used commercial
sources. We aimed to characterize the extent of variability in plasma
proteomes when factors such as freeze–thaw cycles, choice of
anticoagulant, and operator-to-operator performance were accounted
for in the study setup. Plasma samples were analyzed in data-independent
acquisition mode by using two distinct instruments (Exploris 480,
timsTOF HT) and search algorithms (CHIMERYS, DIA-NN). Plasma proteome
enrichment yields ranged from 2.8- to 6.2-fold more compared to the
conventional workflow, achieving yields exceeding 5000 proteins (timsTOF
HT). Notably, the observed variability in proteome composition was
largely attributable to differences in whole blood-to-plasma operating
procedures across commercial sources. While these distinct proteomes
remained undetectable with conventional workflows, particle-dependent
proteome profiling successfully revealed the procedural differences.