Profiles in spectra
Authors:   , Kreft Iris Charlotte 
Published: October 2024

Proteins are key regulators of biological processes and variations in their quantity and quality are related to disease. In this thesis, we use state-of-the-art mass spectrometry (MS) to demonstrate how protein profiling can provide insights into hematological disorders. In chapter 2, MS analysis was employed to explore the effects of whole-blood donation on the abundance of plasma proteins. We demonstrated that MS-based plasma profiling can be successfully employed to detect individual-specific allelic variants (e.g., Alpha-1 antitrypsin) as well as protein trajectories and protein complexes that reflect the functionality and integrity of platelets, red blood cells and immune cells in blood.

In chapter 3, we aimed to investigate whether plasma profiling can aid in identifying and predicting complications related to fever and infection in febrile neutropenia (FN). We performed longitudinal plasma profiling on 26 patients with acute myeloid leukemia (AML) during chemo-induced neutropenia. We revealed correlations between proteins quantified by MS and laboratory measurements, as well as an association between fever- and infection-related complications with a broader inflammatory protein panel.

In chapter 4, we explored the potential of data-independent acquisition (DIA) MS for discovering proteins associated to graft-versus-host disease (aGVHD) in patients with Allogeneic hematopoietic stem cell transplantation (HSCT). We analyzed the plasma proteomes of a small longitudinal cohort of HSCT recipients (n=10). Longitudinal profiling revealed alterations of the proteome after HSCT, most prominently to iron regulation and inflammation-related proteins. Proteomics data were correlated with clinical parameters, resulting in associations between inflammation-related proteins and aGvHD grade.

In chapter 5, we employed MS-based proteomics to investigate the dynamics of protein expression in a ferret model, aiming to elucidate the changes in coagulation and thrombosis associated with COVID-19. We performed plasma profiling on CDV- and SARS-CoV-2-inoculatedand ferrets. We showed the capability MS in identifying protein changes indicative of disease severity. However, our findings revealed only minor variations in plasma protein profiles and no changes in coagulation proteins. This suggests a limited utility of ferrets as an animal model for COVID-19-associated thrombotic complications.

In chapter 6, we advanced our plasma profiling approach using a high-throughput DIA workflow and utilized it in the context of thrombosis. We investigated whether analyzing plasma proteomes across multiple thrombosis cohorts could reveal unique or shared protein profiles linked to thromboembolic events. Changes in plasma proteome profiles were largely attributed to the underlying cause or treatments, as exemplified by decreased levels of vitamin k-dependent proteins in patients receiving vitamin K-antagonists, distinct SARS-COV-2 signatures in COVID-19 patients and inflammatory signatures in patients with CVST.

In chapter 7, we profiled the platelet proteome of patients with inherited platelet disorders (IPDs). We aimed to gain insights into the effects of these pathogenic variants at the protein level, thereby enhancing our understanding of the genotype-phenotype relationship. Platelet proteomes diverged between IPDs, with the most pronounced alterations observed in genes related to cytoskeleton. Moreover, protein correlation analysis revealed known and potential novel alpha granule-associated proteins. Taken together, MS-based protein profiling provided insight into the underlying molecular mechanism of IPDs.

In chapter 8, we employed MS-based protein profiling strategies to bridge the gap between genotyping and functional assays in von Willebrand disease (VWD). We examined VWD patients and uncovered significant heterogeneity in Von Willebrand Factor sequence coverage among patients. Furthermore, we demonstrated the utility of a VWF Variant Database in facilitating proteotyping of VWF variants by identification of pathogenic and benign variants. Using a targeted approach, we were able to accurately identify and quantify VWF proteoforms of increased clearance from blood. Taken together, we showed that MS can be used as a diagnostic assay in patients with Von Willebrand disease to support disease classification.