Highly Reproducible Automated Proteomics Sample Preparation Workflow for Quantitative Mass Spectrometry

Abstract

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 selected reaction monitoring (SRM) assay targeting 6 plasma biomarkers and spiked β-galactosidase, mean intraday and interday cyclic voltammograms (CVs) for 5 serum and 5 plasma samples over 5 days were <20%. In a highly multiplexed SRM assay targeting more than 70 proteins, 90% of the transitions from 6 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. Reproducible quantification of peptides was observed across replicates, days, instruments, and laboratory sites, demonstrating the broad applicability of this approach.

A novel LC system embeds analytes in pre-formed gradients for rapid, ultra-robust proteomics

“…Despite the great technological advances in high sensitivity nano-flow MS-based proteomics, the robustness and throughput have been weak links even in state of the art MS-based proteomic workflows. This has led to a move towards microflow systemsespecially with a view towards clinical applicationshowever, at the cost of sensitivity (Fu et al, 2018). Here, we have introduced an entirely novel concept based on the pre-formation of gradients at relatively high-flow and low-pressure.…”

Systematic Evaluation of Immobilized Trypsin‐Based Fast Protein Digestion for Deep and High‐Throughput Bottom‐Up Proteomics

“…Much effort has been made to improve the throughput of proteomic sample preparation for bottom‐up proteomics in order to facilitate fundamental research and clinical diagnostics . However, hours of tryptic digestion were required in those studies.…”

Developing front‐end devices for improved sample preparation in MS‐based proteome analysis

“…With respect to automating the protein sample preparation process that is integral to MS analysis, researchers recognize the benefits of automation not only to facilitate throughput but also in providing greater precision, particularly with respect to quantitative analysis . Automated liquid handler workstations can now perform the majority of “routine” sample manipulations to process protein samples (in gel or in solution), thus freeing the researcher to focus efforts on more challenging tasks.…”