Comprehensive Analysis of Low-Molecular-Weight Human Plasma Proteome Using Top-Down Mass Spectrometry

While
human plasma serves as a great source for disease diagnosis,
low-molecular-weight (LMW) proteome (<30 kDa) has been shown to
contain a rich source of diagnostic biomarkers. Here we employ top-down
mass spectrometry to analyze the LMW proteoforms present in four types
of human plasma samples pooled from three healthy controls (HCs) without
immunoaffinity depletion and with depletion of the top two, six, and
seven high-abundance proteins. The LMW proteoforms were first fractionated
based on molecular weight using gel-eluted liquid fraction entrapment
electrophoresis (GELFrEE). Then, the GELFrEE fractions containing
up to 30 kDa were subjected to nanocapillary–LC–MS/MS,
and the high-resolution MS and MS/MS data were processed using ProSightPC
3.0. As a result, a total of 442 LMW proteins and cleaved products,
including those with post-translational modifications and single amino
acid variations, were identified. From additional comparative analysis
of plasma samples without immunoaffinity depletion between HCs and
colorectal cancer (CRC) patients via top-down approach, tens of LMW
proteoforms, including platelet factor 4, were found to show >1.5-fold
changes between the plasma samples of HCs and CRC patients, and six
of the LMW proteins were verified by Western blot analysis.