A Practical and Analytical Comparative Study of Gel-Based Top-Down and Gel-Free Bottom-Up Proteomics Including Unbiased Proteoform Detection.

Proteomics is an indispensable analytical technique to study the dynamic functioning of biological systems via different proteins and their proteoforms. In recent years, bottom-up shotgun has become more popular than gel-based top-down proteomics. The current study examined the qualitative and quantitative performance of these two fundamentally different methodologies by the parallel measurement of six technical and three biological replicates of the human prostate carcinoma cell line DU145 using its two most common standard techniques, label-free shotgun and two-dimensional differential gel electrophoresis (2D-DIGE). The analytical strengths and limitations were explored, finally focusing on the unbiased detection of proteoforms, exemplified by discovering a prostate cancer-related cleavage product of pyruvate kinase M2. Label-free shotgun proteomics quickly yields an annotated proteome but with reduced robustness, as determined by three times higher technical variation compared to 2D-DIGE. At a glance, only 2D-DIGE top-down analysis provided valuable, direct stoichiometric qualitative and quantitative information from proteins to their proteoforms, even with unexpected post-translational modifications, such as proteolytic cleavage and phosphorylation. However, the 2D-DIGE technology required almost 20 times as much time per protein/proteoform characterization with more manual work. Ultimately, this work should expose both techniques' orthogonality with their different contents of data output to elucidate biological questions.

Platelet Phenotype Analysis of COVID-19 Patients Reveals Progressive Changes in the Activation of Integrin αIIbß3, F13A1, the SARS-CoV-2 Target EIF4A1 and Annexin A5.

Background: The fatal consequences of an infection with severe acute respiratory syndrome coronavirus 2 are not only caused by severe pneumonia, but also by thrombosis. Platelets are important regulators of thrombosis, but their involvement in the pathogenesis of COVID-19 is largely unknown. The aim of this study was to determine their functional and biochemical profile in patients with COVID-19 in dependence of mortality within 5-days after hospitalization. Methods: The COVID-19-related platelet phenotype was examined by analyzing their basal activation state via integrin αIIbß3 activation using flow cytometry and the proteome by unbiased two-dimensional differential in-gel fluorescence electrophoresis. In total we monitored 98 surviving and 12 non-surviving COVID-19 patients over 5 days of hospital stay and compared them to healthy controls (n = 12). Results: Over the observation period the level of basal αIIbß3 activation on platelets from non-surviving COVID-19 patients decreased compared to survivors. In line with this finding, proteomic analysis revealed a decrease in the total amount of integrin αIIb (ITGA2B), a subunit of αIIbß3, in COVID-19 patients compared to healthy controls; the decline was even more pronounced for the non-survivors. Consumption of the fibrin-stabilizing factor coagulation factor XIIIA (F13A1) was higher in platelets from COVID-19 patients and tended to be higher in non-survivors; plasma concentrations of the latter also differed significantly. Depending on COVID-19 disease status and mortality, increased amounts of annexin A5 (ANXA5), eukaryotic initiation factor 4A-I (EIF4A1), and transaldolase (TALDO1) were found in the platelet proteome and also correlated with the nasopharyngeal viral load. Dysregulation of these proteins may play a role for virus replication. ANXA5 has also been identified as an autoantigen of the antiphospholipid syndrome, which is common in COVID-19 patients. Finally, the levels of two different protein disulfide isomerases, P4HB and PDIA6, which support thrombosis, were increased in the platelets of COVID-19 patients. Conclusion: Platelets from COVID-19 patients showed significant changes in the activation phenotype, in the processing of the final coagulation factor F13A1 and the phospholipid-binding protein ANXA5 compared to healthy subjects. Additionally, these results demonstrate specific alterations in platelets during COVID-19, which are significantly linked to fatal outcome.

Alterations of the Platelet Proteome in Lung Cancer: Accelerated F13A1 and ER Processing as New Actors in Hypercoagulability.

In order to comprehensively expose cancer-related biochemical changes, we compared the platelet proteome of two types of cancer with a high risk of thrombosis (22 patients with brain cancer, 19 with lung cancer) to 41 matched healthy controls using unbiased two-dimensional differential in-gel electrophoresis. The examined platelet proteome was unchanged in patients with brain cancer, but considerably affected in lung cancer with 15 significantly altered proteins. Amongst these, the endoplasmic reticulum (ER) proteins calreticulin (CALR), endoplasmic reticulum chaperone BiP (HSPA5) and protein disulfide-isomerase (P4HB) were significantly elevated. Accelerated conversion of the fibrin stabilising factor XIII was detected in platelets of patients with lung cancer by elevated levels of a coagulation factor XIII (F13A1) 55 kDa fragment. A significant correlation of this F13A1 cleavage product with plasma levels of the plasmin-α-2-antiplasmin complex and D-dimer suggests its enhanced degradation by the fibrinolytic system. Protein association network analysis showed that lung cancer-related proteins were involved in platelet degranulation and upregulated ER protein processing. As a possible outcome, plasma FVIII, an immediate end product for ER-mediated glycosylation, correlated significantly with the ER-executing chaperones CALR and HSPA5. These new data on the differential behaviour of platelets in various cancers revealed F13A1 and ER chaperones as potential novel diagnostic and therapeutic targets in lung cancer patients.

Altered platelet proteome in lupus anticoagulant (LA)-positive patients-protein disulfide isomerase and NETosis as new players in LA-related thrombosis.

Patients with antiphospholipid syndrome (APS) are at high risk of developing venous and arterial thromboembolism (TE). The role of platelets in the pathogenesis of these prothrombotic conditions is not yet fully understood. The aim of this study was to gain mechanistic insights into the role of platelets in APS by comparing the platelet proteome between lupus anticoagulant (LA)-positive patients with (LA+ TE+) and without a history of TE (LA+ TE-) and healthy controls. The platelet proteome of 47 patients with LA, 31 with a history of TE and 16 without thrombotic history, and 47 healthy controls was analyzed by two-dimensional differential in-gel electrophoresis and mass spectrometry to identify disease-related proteins. Afterward, selected LA-related platelet proteins were validated by western blot and ELISA. Alterations of 25 proteins were observed between the study groups. STRING pathway analysis showed that LA-related protein profiles were involved in platelet activation, aggregation, and degranulation. For example, protein disulfide isomerase family members, enzymes that promote thrombosis, were upregulated in platelets and plasma of LA+ TE+ patients. Leukocyte elastase inhibitor (SERPINB1), an antagonist of neutrophil extracellular trap (NET) formation, was decreased in platelets of LA+ TE+ patients compared to healthy controls. Additionally, citrullinated histone H3, a NET-specific marker, was increased in plasma of LA+ TE+ patients. These findings suggest that decreased platelet SERPINB1 levels favor prothrombotic NETosis, especially in LA+ TE+ patients. Our findings reveal protein abundance changes connected to altered platelet function in LA-positive patients, thus suggesting a pathogenic role of platelets in thrombotic complications in APS.