SPAP: Soluble Human Plasma Proteoform Analysis via Acetonitrile Precipitation and Top-Down Mass Spectrometry

Advances in liquid chromatography–mass spectrometry
have
significantly improved proteomic analyses of human plasma. However,
information at the level of intact proteoforms remains limited due
to the high dynamic range of protein abundance and the complexity
of post-translational modifications. To address this challenge, we
introduce soluble plasma proteoform analysis via acetonitrile precipitation
(SPAP), a streamlined workflow for top-down mass spectrometry-based
proteomics that isolates small, intact proteoforms from the acetonitrile-soluble
plasma fraction, enabling direct measurement of proteoform diversity
and post-translational modifications with high resolution. This simple
and scalable method employs cold acetonitrile to precipitate abundant
plasma proteins, thereby enriching the sample for lower-molecular-weight
proteoforms. We first assessed the method’s performance using
a reference plasma sample. To explore its clinical applicability,
we applied SPAP to a cohort of 40 individuals, including 30 patients
with liver cirrhosis and 10 healthy controls. In total, we report
3746 proteoforms from 255 proteins, including those with phosphorylation,
truncation, and disulfide bond modifications. Reproducibility was
confirmed with a coefficient of variation of <10% for the majority
of enriched proteoforms, including those potentially associated with
hemostasis, lipoprotein metabolism, cytoskeletal structure, and protease
regulation. SPAP enabled effective stratification of the three cirrhosis
stages, verifying previously published results and supporting the
identification of candidate biomarkers. Although liver cirrhosis was
used as a model system, the SPAP workflow is broadly applicable to
human disease with proteoform-level resolution, offering a new path
to stronger correlations in smaller cohorts and addressing key challenges
in diagnostic and biomarker discovery.