Rapid and deep plasma proteomics workflows for robust identification and quantification of biomarkers of sickle cell anaemia

Plasma serves as a rich source of protein biomarkers but in-depth proteomic analysis is challenging due to vast dynamic range of protein abundance. Pre-fractionation of plasma proteins is commonly practiced to enhance the proteome coverage but the protocols are time-expensive, suffer from flowchart complexity, and often less reproducible. Here, we explore multiple strategies of shotgun proteomics to optimize biomarker discovery workflows for Sickle Cell Anaemia (SCA) patients from Maharashtra, India. A deep proteomics workflow via off-line reverse phase ultra-high-pressure liquid chromatography-based fractionation of tryptic digested plasma peptides followed by optimized pooling of peptides based on charge and hydrophobicity yielded the best depth of plasma proteome with a trade-off of significantly long experimental time. Alternatively, a rapid analysis of tryptic digested plasma peptides via a shorter gradient mass spectrometry run saves time but quantifies only ~ 50% of the proteins than the deep workflow. Intriguingly, despite the difference in proteome coverage, more than 80% of known FDA and SCA biomarkers quantified in the deep workflow are also quantified in the rapid workflow. Given the practical difficulties of sample collection and plasma preservation in rural India, we propose the deep proteomics workflow for biomarker discovery in smaller cohorts and the rapid workflow for biomarker validations in larger cohorts. Targeted-proteomics-based strategies may be designed for the validation of missing biomarkers in the rapid workflow.