Plasma Glycoproteins and Biotherapeutics from a Proteoform Perspective

Dario Alexander Taco Cramer

Research output: ThesisDoctoral thesis 1 (Research UU / Graduation UU)

Abstract

For an analytical chemist interested in plasma proteins, a key question is: how do we best analyze (glyco)proteins and their glycans in all their beauty? This thesis describes an approach to these questions using peptide-centric and native mass spectrometry. A thorough introduction of protein characteristics on a biomolecular level, mass spectrometry techniques and glycosylation is followed by a review of current cell expression systems for recombinant glycoproteins. Using a combination of ion-exchange chromatography and native mass spectrometry, blood serum proteins are investigated in depth, revealing a wealth of proteoforms of serum glycoproteins, including new annotations such as phosphorylation and specific glycoforms, spanning a large range of masses. The metalloprotein cerulopasmin was also characterized including its copper metal masses by combining several analytical techniques. Additionally, two serum glycoproteins are studied in detail – alpha-1-antitrypsin and plasminogen. In the first, common mutations were resolved by native mass spectrometry with such detail that it was possible to quantify the different allelic expressions, revealing a discrepancy in abundance between common allelic mutations. In the latter, a new intermediate step in the activation of plasminogen to plasmin was found, as well as its elusive phosphorylation site by combining peptide-centric and native mass spectrometry. Knowledge gained on the analysis of these serum glycoproteins was also applied to complex, high molecular weight antibodies and their many by-products, making it possible to characterize potentially unwanted by-products on the molecular level. Here, the use of size-exclusion chromatography combined with mass photometry under various conditions permitted to draw conclusions on the effect of different by-products and their stability.
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Utrecht University
Supervisors/Advisors
  • Heck, Albert, Supervisor
  • Reusch, D., Co-supervisor, External person
Award date30 Sept 2024
Place of PublicationUtrecht
Publisher
Print ISBNs978-94-6506-425-3
DOIs
Publication statusPublished - 30 Sept 2024

Keywords

  • serum glycoproteins
  • mass spectrometry
  • glycosylation
  • recombinant proteins
  • native mass spectrometry
  • proteoform profiling

Fingerprint

Dive into the research topics of 'Plasma Glycoproteins and Biotherapeutics from a Proteoform Perspective'. Together they form a unique fingerprint.

Cite this