Differentially Expressed Proteins in Transfusion-Dependent Thalassemia (TDT) and Their Role in Tdt Disease Severity

Introduction: The management of transfusion-dependent thalassemia (TDT) has seen essential improvements; however, still several challenges to controlling the incidence worldwide. Limitations of available current treatment modalities in resource-limited countries are the driving force to investigate the novel diagnostic and therapeutic options for better clinical management of these patients. Proteomics may be a potential technique to identify novel biomarkers of disease severity and understand the pathophysiological mechanisms of TDT, as proven in many other diseases. Understanding the inter-relationships of proteins affected by the disease process, such as oxidative stress in TDT patients, can help to elucidate the mechanisms involved in the disease pathophysiology. However, plasma proteome profile studies in TDT are limited. This study aimed to investigate the differentially expressed proteins (novel proteins) in serum from TDT to understand the pathophysiology of TDT and determine their role in TDT severity by performing quantitative serum proteomics profiling.

Methods: The sample size was calculated based on the sensitivity and specificity of the protein biomarkers identified in the pilot study. A total of 64 patients were included in this study. A serum sample was collected from 32 TDT patients and 32 non-thalassemia controls. Before the serum samples could be processed for label-free quantification using the liquid chromatography-mass spectrometry/mass spectrometry (LCMS/MS) approach, high abundance proteins such as serum albumin and immunoglobulins were depleted using Pierce™ Albumin/IgG Removal Kit (Thermo Fisher Scientific, USA.) according to the manufacturer's protocols. The protein content in samples after depletion of high abundance proteins was determined using the Bradford assay using the protocol recommended by the manufacturer protocol (BioRad, USA). Each protein sample was prepared for LC-MS/MS analysis using EasyPep™ Mini MS Sample Prep Kit (Thermo Fisher Scientific, USA). Analysis of sera was performed on the Agilent 6560C ion mobility Q-TOF LC/MS system (Agilent, USA). To validate the LCMS/MS results, ELISA was done. Differentially regulated proteins with p-value <0.05 were identified from the dataset and uploaded to online databases PANTHER to classify and identify the proteins' functions. The functional gene ontology (GO) annotation of the identified protein in terms of molecular function, biological process, and cellular component was evaluated using this curated biological database. The statistically significant expressed proteins were submitted to STRING, and a combined score of >0.4 (medium confidence) was considered experimentally validated and statistically significant interactions between proteins. The cluster with the largest number of proteins with strong connections was identified for the pathway enrichment analysis. The identified protein cluster from the STRING database was uploaded to the KEGG online database to acquire the enriched pathway.

Results: 51 dysregulated proteins were found between cases and controls (Table). However, only 13 showed statistically significant differential expression (p <0.05) with a fold change >1, and mostly were downregulated. Gene ontology functional annotation analysis showed these proteins were involved in molecular functions, mainly catalytic activities (37%), followed by molecular function regulation (15%). The biological processes primarily involved the cellular (35.5%) and metabolic processes (32.3%). The pathway analysis showed that these proteins were involved mainly in hemostasis. In KEGG pathway analysis, the proteins were involved primarily in fat digestion and absorption and vitamin digestion & absorption.

Conclusion: We elucidated the possible biological processes modulated by oxidative stress using the differentially expressed novel proteins and the associated pathways using comprehensive molecular and biocomputational analysis. Interestingly, most of the differentially expressed proteins in this study have not been yet reported in previous studies to be modulated by oxidative stress in TDT patients. Therefore, the identified dysregulated proteins can serve as a potential biomarker to monitor a patient's condition, determine the effect, need for blood transfusion, and help develop novel prognostic and diagnostic methods.

No relevant conflicts of interest to declare.