The discovery of kidney injury-related protein biomarkers associated with first-line anteretroviral treatment in South Africa using microflow SWATH mass spectrometryre
Date
2019
Authors
Govender, Ireshyn Selvan
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Abstract
Background: South African patients account for ~10% of the global HIV/AIDS burden. In response, the country has implemented the largest antiretroviral therapy (ART) program in the world. However, not all patients respond positively, and it has been observed that up to 8-10% of patients on first-line ART experience acute kidney injury (AKI). Current tests for AKI are unreliable and only reflect positive following significant kidney damage. Hence there is a dire need to identify biomarkers for early detection of AKI. The urinary proteome provides a large and diverse source of information pertaining to a patient’s health status. To date, there is no - published - standard operating protocol for reproducible collection and processing of urine for mass spectrometry-based clinical proteomics.
Methods: Extensive workflow development for urinary proteome analysis was done including multiple protein extraction, liquid chromatography and mass spectrometry acquisition methods. Acetone precipitation, of urinary protein, followed by FASP was compared to two novel sample preparation techniques using MagReSyn® HILIC and Norgen Biotek Corporation Urine Kits. The kit-based method was applied to a clinical cohort of 74 HIV positive patients. Their urinary proteomes were compared with the aim of detecting markers associated with AKI due to first-line ART using SWATH-MS.
Results and Discussion: Using MagReSyn® HILIC was the easiest, fastest and cheapest method whilst generating the most comprehensive data. The kit-based method was the most easily implemented at the point-of-care clinic and generated data that was comparable to the MagReSyn® HILIC method. Using the kit-based method, this study identified many proteins (≥ 4-fold change, q value ≤ 0.01, ≥ 2 unique peptides), previously reported in literature, that have an association with kidney dysfunction. Furthermore, novel, putative markers for AKI have been identified including mimecan which has not been reported as an AKI marker before. Additionally, proteins showing significant changes in urinary abundance allude to the process of renal hepatisation since many of these proteins are usually associated with the liver. Some of these proteins have not been reported in relation to AKI and have not been reported in any South African populations before.
Conclusion: The methods developed in this study can serve as the gold-standard for future urinary proteomics experiments. Additionally, following assessment in a blinded verification cohort, previously reported and novel proteins have the potential to be used as early markers for AKI.
Description
A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg in fulfillment of the requirements for the degree of Doctor of Philosophy
January 2019