Developing a Proteomic Signature for the Early Detection of Heart Failure in Type 2 Diabetics

The prevalence of Type 2 Diabetes (T2D) globally exceeds 500 million individuals, constituting a significant health concern. T2D patients are at increased risk of developing heart failure (HF) by at least 2-fold. Therefore, this PhD aims to identify protein biomarkers from plasma, using Liquid Chromatography tandem mass spectrometry capable of predicting early-stage HF within T2D patients. The clinical variables: global longitudinal strain (GLS), the ratio of early diastolic mitral inflow velocity (E) to early diastolic mitral annulus velocity (e’) (E/e’), extracellular volume fraction (ECV) and peak volume of oxygen consumption (VO2 max) were used as variables predictive of HF. The overall aim can be split into 3 sub-objectives: the identification of candidate biomarkers using high-resolution MS, the development of a multiple reaction monitoring (MRM) assay for the quantification of proteotypic peptides representing target proteins, and the utilisation of this assay to conduct a verification study of identified biomarkers.

To achieve these objectives, a biomarker discovery study was undertaken on 96 participants recruited as part of the PREDICT study consisting of 69 T2D patients. Plasma underwent enrichment for extracellular vesicles and lipid-associated proteins via binding with lipid removal agent. Bottom-up proteomics was performed on the resulting proteins. Novel data processing techniques were applied to remove unwanted variation by employing surrogate variable analysis where, dummy variables are created that represent the variation in the data not attributable to the variable of interest. The normalised data were correlated against the four variables of interest to determine which proteins were associated with early HF.

A crude custom peptide library facilitated the identification of proteotypic peptides for the 27 candidate biomarkers identified during the discovery study. A total of 42 peptides were identifiable in a crude plasma digest, and an MRM assay was created to quantify these peptides. Sixteen peptides pass the bioanalytical method validation and were implemented in the clinical verification study. Analysis of the verification study revealed three peptides from three distinct proteins exhibited a significant association with VO2 max.