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 language | English |
---|---|
Qualification | Doctor of Philosophy |
Awarding Institution |
|
Supervisors/Advisors |
|
Award date | 30 Sept 2024 |
Place of Publication | Utrecht |
Publisher | |
Print ISBNs | 978-94-6506-425-3 |
DOIs | |
Publication status | Published - 30 Sept 2024 |
Keywords
- serum glycoproteins
- mass spectrometry
- glycosylation
- recombinant proteins
- native mass spectrometry
- proteoform profiling