Sample preparation for protein quantification by mass spectrometry requires multiple processing steps including denaturation, reduction, alkylation, protease digestion, and peptide cleanup. Scaling these procedures for the analysis of numerous complex biological samples can be tedious and time-consuming, as there are many liquid transfer steps and timed reactions where technical variations can be introduced and propagated. We established an automated sample preparation workflow with a total processing time for 96 samples of 5 h, including a 2 h incubation with trypsin. Peptide cleanup is accomplished by online diversion during the LC/MS/MS analysis. In a highly multiplexed SRM assay targeting more than 70 proteins, 90% of the transitions from plasma samples repeated on 3 separate days had total CVs below 20%. Similar results were obtained when the workflow was transferred to a second site: 93% of peptides had CVs below 20%. An automated trypsin digestion workflow yields uniformly processed samples in less than 5 h. In addition, we developed fully automated peptide Solid phase extraction (desalting) workflow for liquid chromatography-tandem mass spectrometry analysis using a liquid handler workstation with an average CV of 10-20%.  Reproducible quantification of peptides was observed across replicates, days, instruments, and laboratory sites, demonstrating the broad applicability of this approach. This automation thus provides opportunities for high-throughput proteomics research that can result in global proteomic analysis and biomarker discovery, quantification in large clinical sample size settings.

Highly Reproducible Automated Proteomics Sample Preparation Workflow for Quantitative Mass Spectrometry with Accuracy

ACS Spring Meeting 2021

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