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Clotting disorders and wound healing represent a significant health concern to a growing population advancing in age. Fibrinogen, a glycoprotein complex, is a critical molecule in the clotting process but has historically been difficult to analyze at the molecular level. Upon activation, fibrinogen forms large fibrin biopolymers that coalesce into clots that assist in wound healing. However, limited insights into their molecular architecture, due to the sheer size and the insoluble character of fibrin clots, have restricted our ability to develop novel treatments for clotting diseases. Investigating this architecture can currently only be achieved through cross-linking mass spectrometry. In this technique, cross-linking reagents are used to covalently link amino acids in close proximity. This results in peptide pairs after proteolytic digestion that are typically present at low abundance in the background of normal peptides and single peptides linked to the reagent (mono-link). To a large degree, the analytical challenge can be overcome by using enrichable cross-linking reagents, where an enrichment handle is incorporated on the reagent, focusing on peptides covalently linked to the reagent (cross-links & mono-links). The overwhelming complexity of fibrin clots, however, requires an extra enrichment step. In addition, ion mobility separates molecules in the gas phase, providing information about their size as collisional cross-sections.

In this GEN webinar, our expert panelists will discuss how the timsTOF Pro mass spectrometry (MS) platform combines trapped ion mobility with a quadrupole, collision cell, and a TOF mass analyzer to probe ions at high speeds with on-the-fly fragmentation. Moreover, we will also learn how ion mobility is beneficial for cross-linking MS on this platform by enabling the separation of mono-linked peptides from cross-linked peptide pairs. Finally, our panelists will show that with this technique, they were not only able to uncover the structural organization of fibrin clots but also the elusive binding partner FXIII, an enzyme that transiently interacts with fibrin and is involved in forming natural cross-links inside the clot to strengthen its conformation.

A live Q&A session followed the presentations, offering a chance to pose questions to our expert panelists.

Richard Scheltema, PhD
Richard Scheltema, PhD
Assistant Professor, Pharmaceutical Sciences
Utrecht University
Gary Kruppa, PhD
Gary Kruppa, PhD
Vice President of Proteomics
Bruker Daltonics

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