Quantitative proteomics of plasma vesicles identify novel biomarkers for hemoglobin E/β-thalassemic patients

Abstract

Key Points• Chaperones, antioxidants, ironsequestering proteins, and cathepsin S exhibited increased abundance in thalassemic EVs.• Haptoglobin and hemopexin are reduced in thalassemic patients' EVs, reflecting hemolysis. These could be used as clinical biomarkers.Hemoglobin E (HbE)/b-thalassemia has a wide spectrum of clinical manifestations that cannot be explained purely by its genetic background. Circulating extracellular vesicles (EVs) are one factor that likely contributes to disease severity. This study has explored the differences in protein composition and quantity between EVs from HbE/b-thalassemic patients and healthy individuals. We used tandem mass tag labeling mass spectrometry to analyze the EV proteins isolated from the plasma of 15 patients compared with the controls.To reduce biological variation between individuals, the EV proteins isolated from randomly assigned groups of 5 HbE/b-thalassemic patients were pooled and compared with 5 pooled age-and sex-matched controls in 3 separate experiments. Alpha hemoglobin-stabilizing protein had the highest fold increase. Catalase, superoxide dismutase, T-complex proteins, heat shock proteins, transferrin receptor, ferritin, and cathepsin S were also upregulated in thalassemic circulating EVs. Importantly, haptoglobin and hemopexin were consistently reduced in patients' EVs across all data sets, in keeping with the existing hemolysis that occurs in thalassemia. The proteomic data analysis of EV samples isolated from 6 individual HbE/b-thalassemic patients and western blotting results corroborated these findings. In conclusion, we have successfully identified consistent alterations of protein quantity between EVs from HbE/b-thalassemic and healthy individuals. This work highlights haptoglobin, hemopexin, and cathepsin S as potential clinically relevant biomarkers for levels of hemolysis and inflammation. Monitoring of these plasma proteins could help in the clinical management of thalassemia.

Proteomics of Extracellular Vesicles: Update on Their Composition, Biological Roles and Potential Use as Diagnostic Tools in Atherosclerotic Cardiovascular Diseases

“…An additional separation step of the extracted EV proteins is usually performed before MS analysis using either gel electrophoresis [ 125 ] or LC [ 126 , 127 ] to facilitate proteomic analysis removing the contaminants in the sample. Moreover, specific separation methods for the study of post-translational modifications of EV proteins have also been applied in many proteomic studies.…”

“…Quantitative MS based on label and label-free approaches has been applied to study EV proteome in several diseases. Label-free approaches are simple and cost-efficient techniques that have been extensively used in EVs research [ 132 , 133 , 134 ], while label-based approaches include a metabolic or chemical labelling which provides more accurate protein quantification [ 127 , 135 ].…”

Red Blood Cell Homeostasis: Mechanisms and Effects of Microvesicle Generation in Health and Disease

“…This model will benefit from the comparison of the composition and characteristics of RBC-derived microvesicles with microvesicles generated by other cell types, and with exosomes. RBC-derived microvesicles are potentially sensitive and specific biomarkers for the clinical severity of RBC-centered diseases such as sickle cell disease, thalassemia or spherocytosis ( Reliene et al, 2002 ; Hebbel and Key, 2016 ; Kittivorapart et al, 2018 ). Also, microvesicles may reveal the activity as well as the clinical consequences, such as anemia or thrombosis, of systemic processes, such as inflammation.…”

Application of Extracellular Vesicles Proteomics to Cardiovascular Disease: Guidelines, Data Analysis, and Future Perspectives

“…The main labeling strategy used for characterizing circulating EVs is the isobaric tags for relative and absolute quantitation (iTRAQ) . However, some groups also used another labeled alternatives such as isotope‐coded affinity tag (ICAT) or tandem mass tags (TMT) …”