Introduction: Targeted mass spectrometry (MS) can be used for sensitive quantification of specific proteins in complex biological samples. By selectively monitoring predefined peptide ions, targeted MS enables accurate measurement of protein abundance, aiding biomarker discovery and development. However, it is crucial to analytically validate targeted MS assays to ensure their reproducibility. Analytical validation includes assessment of the stability of the assay, ensuring that the results are consistent over time. Stability is essential for the practical application of an assay.

We aim to use targeted MS to measure proteins associated with high-density lipoprotein particles (HDL-P), to develop and validate a biomarker-based risk stratification platform for assessing metabolic health in individuals with obesity.

Methods: A high-throughput method for HDL isolation has been developed. Proteotypic peptide assays for proteins of interest, related to metabolic health in people with or without obesity, were developed. The protein panel for multiple reaction monitoring (MRM) assays was selected based on literature searches, and in-house discovery proteomics experiments. MRM peptide analysis was performed using an Agilent 6490 triple-quadrupole MS with Jet-Stream electrospray source coupled to a 1290 Quaternary Pump HPLC system. The number of transitions used for assay development included all b and y ions, with the final number of transitions per peptide reduced to 3-4 based on characteristics such as intensity and peak shape. Peptide retention times (RT) and optimized collision energies were investigated using Skyline software (Version 23.1; MacCoss Lab) to establish a dynamic MRM-scheduled method based on input parameters of 600ms cycle times and 2-minute RT windows.

Results: An MRM method has been developed to measure 82 proteins using 196 peptide MRM assays, represented by 596 transitions with a gradient time of 36 minutes. The reproducibility of multiple sample injections of the same HDL-P sample resulted in an overall coefficient of variation (CV) of less than 10%. The peptides associated with ApoA-1, the most abundant HDL-associated protein, had a CV <20%. Investigating the HDL-P associated peptides measured over a time course of 5 days showed minimal differences, indicating that HDL associated peptides are stable for at least 120hrs.

Conclusions: The findings demonstrate that MRM can measure HDL-P associated proteins with high reproducibility, and show stability for up to 5 days. Upon completion of additional analytical validation experiments (within-day variability; and intra/inter operator), samples from patients will be analysed to delineate people with obesity high-risk, who are at high-risk of developing life-limiting complications from those who are low-risk and who can maintain their metabolic health despite excessive adiposity.