Sequential Precipitation and Delipidation Enables Efficient Enrichment of Low-Molecular Weight Proteins and Peptides from Human Plasma

Low-molecular weight
proteins and peptides (LMWPs, <30 kDa)
in human plasma serve as potential biomarkers or drug targets and
are endowed with desirable traits for biological and clinical studies.
However, the identification of LMWPs from plasma is retarded by high-abundance
proteins, high-molecular weight proteins, and lipids. Here, we present
a sequential precipitation and delipidation (SPD) method for the efficient
enrichment of LMWPs based on methyl-tert-butyl ether/methanol/water
systems. The enriched LMWP sample was analyzed by single-shot liquid
chromatography–tandem mass spectrometry employing both HCD
and EThcD without tryptic digestion, and 725 peptides were identified
on average. The LMWP sample was also digested and analyzed using a
bottom-up proteomics pipeline, and 289 proteins were identified, of
which 129 (44.6%) proteins were less than 30 kDa and lipoprotein-associated
proteins were significantly enriched. Additionally, 25 neuropeptides
and 19 long noncoding RNA-encoded polypeptides were identified. Taken
together, the SPD method shows good sensitivity and reproducibility
when compared with other enrichment methods and has great potential
for clinical biomarker discovery and application.