Charting the Proteoform Landscape of Serum Proteins in Individual Donors by High-Resolution Native Mass Spectrometry

Most proteins in serum are glycosylated, with several
annotated
as biomarkers and thus diagnostically important and of interest for
their role in disease. Most methods for analyzing serum glycoproteins
employ either glycan release or glycopeptide centric mass spectrometry-based
approaches, which provide excellent tools for analyzing known glycans
but neglect previously undefined or unknown glycosylation and/or other
co-occurring modifications. High-resolution native mass spectrometry
is a relatively new technique for the analysis of intact glycoproteins,
providing a “what you see is what you get” mass profile
of a protein, allowing the qualitative and quantitative observation
of all modifications present. So far, a disadvantage of this approach
has been that it centers mostly on just one specific serum glycoprotein
at the time. To address this issue, we introduce an ion-exchange chromatography-based
fractionation method capable of isolating and analyzing, in parallel,
over 20 serum (glyco)­proteins, covering a mass range between 30 and
190 kDa, from 150 μL of serum. Although generating data in parallel
for all these 20 proteins, we focus the discussion on the very complex
proteoform profiles of four selected proteins, i.e., α-1-antitrypsin,
ceruloplasmin, hemopexin, and complement protein C3. Our analyses
provide an insight into the extensive proteoform landscape of serum
proteins in individual donors, caused by the occurrence of various N- and O-glycans, protein cysteinylation,
and co-occurring genetic variants. Moreover, native mass intact mass
profiling also provided an edge over alternative approaches revealing
the presence of apo- and holo-forms of ceruloplasmin and the endogenous
proteolytic processing in plasma of among others complement protein
C3. We also applied our approach to a small cohort of serum samples
from healthy and diseased individuals. In these, we qualitatively
and quantitatively monitored the changes in proteoform profiles of
ceruloplasmin and revealed a substantial increase in fucosylation
and glycan occupancy in patients with late-stage hepatocellular carcinoma
and pancreatic cancer as compared to healthy donor samples.