Standardized workflow for precise mid- and high-throughput proteomics of blood biofluids

Abstract

Background: Accurate discovery assay workflows are critical for identifying authentic circulating protein biomarkers in diverse blood matrices. Maximizing the commonalities in the proteomic workflows between different biofluids simplifies the approach and increases the likelihood for reproducibility. We developed a workflow that allows flexibility for high and mid–throughput analysis for three blood–based proteomes: naive plasma, plasma depleted of the 14 most abundant proteins, and dried blood. Methods: Optimal conditions for sample preparation and DIA–MS analysis were established in plasma then automated and adapted for depleted plasma and whole blood. The MS workflow was modified to facilitate sensitive high–throughput or deep profile analysis with mid–throughput analysis. Analytical performance was evaluated from 5 complete workflows repeated over 3 days as well as a linearity analysis of a 5—6–point dilution curve. Result: Using our high-throughput workflow, 74%, 93%, 87% of peptides displayed an inter-day CV<30% in plasma, depleted plasma and whole blood. While the mid-throughput workflow had 67%, 90%, 78% of peptides in plasma, depleted plasma and whole blood meeting the CV<30% standard. Lower limits of detection and quantitation were determined for proteins and peptides observed in each biofluid and workflow. Combining the analysis of both high–throughput plasma fractions exceeded the number of reliably identified proteins for individual biofluids in the mid–throughput workflows. Conclusion: The workflow established here allowed for reliable detection of proteins covering a broad dynamic range. We envisage that implementation of this standard workflow on a large scale will facilitate the translation of candidate markers into clinical use.

The autoimmune signature of hyperinflammatory multisystem inflammatory syndrome in children

“…The autoimmune signature of hyperinflammatory multisystem inflammatory syndrome in children discovery proteomics analysis on native and depleted (top 14 most abundant proteins) serum or plasma samples (ref. 21, Figure 1B, and Supplemental Figure 2A). Both data sets were integrated at the transition, peptide, and protein level.…”

“…Detailed proteomics methods can be found in the supplemental material and are outlined in McArdle et al (21).…”

“…Digestion. Plasma/serum and depleted plasma/serum trypsin digestion and desalting were performed using an automated workstation (Beckman i7), which is programed to perform reactions at a controlled temperature with uniform mixing, as previously described (61) using a standardized sample processing workflow (21).…”

Automation of high-throughput mRNA-seq library preparation: a robust, hands-free and time efficient methodology

“…[17][18][19][20] Other omics platforms where automation of the sample preparation workflows has been demonstrated include proteomic sample preparation needed for liquid chromatography with mass spectrometry (LC-MS/MS) data analysis. 21,22 Automation of library preparation for RNA sequencing is an area of active research and investigation.…”

Automated proteomic sample preparation: The key component for high throughput and quantitative mass spectrometry analysis

“…This clinical assay utilizes solid phase extraction-based automated workflow on a Hamilton automation liquid handler following trypsin digestion of CSF (DeMarco et al, 2020;Weber et al, 2019). Our group have integrated a positive pressure apparatus for desalting on HLB plates using the i7 hybrid liquid handling workstation (Beckman Coulter) (Johnson et al, 2020;McArdle et al, 2021). In our lab, the liquid handling %CV was calculated to be around 6% (when the desalting step was included, the total %CV was ~10%; and without desalting the total %CV was ~4%) (Figure 3 bottom right panel).…”