Proteomics Goes 4D to Enhance Understanding of Clotting Diseases
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 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.
Presenter: Gary Kruppa, Ph.D. (Vice President Proteomics at Bruker Daltonics Inc., and Managing Director of Bruker S.R.O., Billerica, MA, USA)
Gary Kruppa serves as the Vice President for Proteomics at Bruker Daltonics Inc. He manages a growing global team of applications, field applications and applications development scientists, focused on developing new proteomics applications for the timsTOF Pro, and supporting customers and collaborators. Dr. Kruppa earned his bachelor’s degree in chemistry at the University of Delaware and his Ph. D. in chemical physics at CalTech. Dr. Kruppa has been working with Bruker since 1991, where he first worked on the FTMS product line, later serving as Vice President for FTMS. From 2001 to 2004, Dr. Kruppa ran his own research lab at Sandia National Labs, where he and his team developed novel methods and reagents for chemical crosslinking of proteins for the study of protein structure. In 2004 he rejoined Bruker, where he served as Vice President for Business Development until 2014. From 2014 to 2016 Dr. Kruppa worked as CEO of MRM Proteomics Inc., to commercialize kits for quantitative MRM proteomics analysis of potential biomarkers. Dr. Kruppa returned to Bruker in 2016 to serve as Vice President for proteomics, where he helped launch the revolutionary timsTOF Pro QTOF Mass Spectrometer powered by the PASEF method.
Presenter: Richard Scheltema, Ph.D. (Assistant Professor, Pharmaceutical Sciences, Utrecht University, Utrecht, Netherlands)
Richard currently runs his own, independent research group at Utrecht University. After finalizing his PostDoc working on LC-MS/MS platforms within the group of Prof. Dr. Matthias Mann (Max Planck Institute of Biochemistry, DE), he started the Scheltema laboratory at Utrecht University. The focus lies on structural proteomics applied to medium and high complexity protein environments like the ECM and membrane proteins. Mass spectrometry based approaches are employed that are independent of protein size, deal with high complexity mixtures and play well with other structural biology techniques like Cryo-EM and crystallography to uncover novel and exciting biology. The technological developments and their applications have been published in journals like Nature Protocols, Nature Communications, Molecular Cell, Cell Reports, ACS Central Science, and PNAS and additionally have been commercialized and are worldwide in use.
