High throughput abundant protein depletion for plasma proteomics

Biological fluids like plasma, etc. are widely used for proteomics-based disease biomarker discovery. However, its analysis is challenging due to the complexity and wide dynamic range of the proteome, ranging over 10 orders of magnitude. The high abundant proteins like albumin, IgGs etc. constitute about 90% of the plasma and their presence masks the identification of the low abundant proteins in the sample. Several workflows have been developed around depleting these abundant proteins to enable the deep penetration into the proteome to identify low abundant proteins of interest. These workflows are laborious with multi-step protocols which greatly increases hands on- time while processing multiple sample types. To address these issues, we developed a 96 well plate format of the depletion resin to support higher sample throughput. This format can deplete 96 samples at a time with a depletion efficiency of ≥ 95% and showed similar performance to our spin column format in terms of protein/peptide identifications and Top14 abundant proteins depletion in the plasma samples improving the proteome coverage by 2.4-fold. We have also evaluated our format with different biological fluids, such as CSF etc. In addition, our depletion resin in 96 well format combined with our optimized EasyPep chemistry can process these samples in 7-8 hours with high reproducibility and <5% CVs. These two combined can significantly eliminate the hands-on-challenges while handling a large number of samples. Moreover, combining our depletion resin in plate format with EasyPep96 magnetic using an automated KingFisher platform generated ready-to-inject samples in less amount of time and showed similar protein/peptide identifications with ≥ 95% depletion efficiency compared to other workflows. Differentially expressed proteins in a cancer vs. normal human plasma samples were identified and quantified using a label-free data-independent acquisition (DIA) strategy. Overall, we demonstrate that our depletion resin in a 96 well format enables the depletion of Top14 abundant proteins from the biological fluids with a greater depletion efficiency for high throughput plasma proteomics.