Charting the proteoform landscape of serum proteins in individual donors by high-resolution native mass spectrometry

ABSTRACT: 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 have used 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 IEX-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. The proteoform profiles of four selected proteins of interest, i.e., alpha-1-antitrypsin, ceruloplasmin, hemopexin and complement protein C3, are characterized and discussed in-depth. Our detailed analyses enabled the annotation of N- and O-glycans, protein cysteinylation, metal-ion binding, endogenous proteolysis and the detection of co-occurring genetic variants. Finally, we applied our approach to a small set of serum samples from healthy and diseased individuals. In these, we qualitatively and quantitatively monitored the changes in proteoform profiles of ceruloplasmin and reveal a substantial increase in fucosylation and glycan occupancy in patients with late-stage hepatocellular carcinoma and pancreatic cancer as compared to healthy donor samples. 

INSTRUMENT(S): Q Exactive Plus, Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Plasma

SUBMITTER: Dario Cramer  

LAB HEAD: Albert Heck

PROVIDER: PXD033978 | Pride | 2022-09-06

REPOSITORIES: Pride