Standardized workflows for precise high-throughput proteomics of blood biofluids
Standardized workflows for precise high-throughput proteomics of blood biofluids
Innovations in proteomics methodologies have emerged in recent years, but there are still obstacles inhibiting the translation of biomarkers into clinical use. Streamlined preanalytical proteomic workflows that achieves sensitive and robust analysis of multiple complex blood-based matrices with sufficient reliability for large population-based screening are therefore needed.
A new paper by the Van Eyk group from Cedars-Sinai Medical Center in Los Angeles describes the development of such a workflow for three blood-based proteomes: naive plasma, plasma depleted of the 14 most abundant proteins, and whole blood utilizing the 60 samples per day methods on the Evosep One coupled to a Thermo Orbitrap Exploris 480 MS.
Commercially available biofluids were subjected to protein denaturation, reduction, alkylation and digestion using a Beckman Biomek i7 automated workstation. Initially, they assessed the impact of temperature and time on trypsin proteolytic efficiency and found a 4-hour digestion at 42°C in the presence of 5% TFE to be most suited for maximizing throughput and efficiency.
Optimal parameters for DIA acquisition parameters for plasma
To ensure reproducibility throughout the entire workflow, they also established optimal conditions for DIA analysis in plasma and adapted this to depleted plasma and whole blood. They tested eight different MS methods covering four isolation window widths and two resolution settings, as the instrument cycle time is influenced by resolution settings and number of mass windows which in turn impact the number of identifications and reproducibility.
They found the number of protein identifications across all parameters to be comparable with nearly 200 proteins identified. While one method supported the highest number of peptide identifications compared to the other methods, it performed worst in terms of reproducibility on protein level. Thus, they decided to base their workflow on the method providing the highest number of quantified peptides with a coefficient of variation below 30%, namely the 21 Da method and 50 windows with a resolution of 15,000. Importantly, data acquired using their method were associated with more than 8 data points across the curve, a well-established benchmark for acceptable quantification.
Precision of standardized workflow
They applied their optimized protocol and compared the number of identifications along with the intra- and inter-day reliability of the workflow on three commonly analyzed biofluids: naive plasma, depleted plasma, and whole blood. To determine the analytical precision, five replicate samples of each biofluid were digested once a day for three consecutive days and analyzed. This analysis revealed that most peptides were identified with a coefficient of variation below 30% on individual days. Comparing across all three days, plasma showed the greatest drop in peptide intensity reproducibility with only 74% of all peptides achieving a coefficient of variation below 30%, compared to 93% and 87% of peptides in depleted plasma and blood, respectively.
Collectively, this paper describes the development of a streamlined process of blood biofluids covering a broad dynamic range associated with minimal hands-on time, fast turnaround, and excellent intra- and inter-day stability. Interestingly, the inter-day precision of depleted plasma was the most stable with more than 90% of peptides reproducibly detected across days. The implementation of standardized workflows like this on a large scale will facilitate the translation of candidate markers into clinical use.
Link: https://www.biorxiv.org/content/10.1101/2021.03.26.437268v1.full.pdf
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