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.

Keywords: automation; high throughput; liquid chromatography selected reaction monitoring; liquid chromatography-tandem mass spectrometry; protein sample preparation; reproducibility.

Figure 1.. Automated proteomic sample preparation schema.

A) Workflow and layout of the automated laboratory workstation. B) Outline of the automated digestion protocol. See Materials and Methods for details.

Figure 2.. Reproducibility of the automated proteomic sample preparation workflow with a highly multiplexed SRM analysis.

Six plasma aliquots were processed three times on separate days. The resulting peptides were analyzed by LC MS/MS with a 30-minute LC gradient and a scheduled SRM method targeting 72 proteins. A) Average intensities (left) and inter-day % CVs (right) of the three independent digests (see supplemental table 5). B) Results stratified by average signal intensity.

Figure 3.. Trypsin digestion time course.

Pooled human plasma was digested with trypsin for various times and then analyzed with the highly multiplexed SRM assay. Recoveries for each peptide were normalized to the 2-hour time-point. A) Results for 162 peptides from 70 proteins are plotted individually (left) and collectively (right). B) Peak intensity variations over time for peptides from albumin, alpha-1 antitrypsin, and alpha-1-antichymotrypsin.

Figure 4.. Inter-lab reproducibility.

The final automated processing method was implemented at Cedars Sinai Medical Center (site 1, n=24) and at SCIEX (site 2, n=48). SRM assay results are presented for 360 transitions from 93 peptides representing 44 proteins that were measured at both sites. A) % CVs for individual peptides. B) Results stratified by % CV ranges.