Highly Reproducible Automated Proteomics Sample Preparation Workflow for Quantitative Mass Spectrometry

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.