Comprehensive and Scalable Highly Automated MS-Based Proteomic Workflow for Clinical Biomarker Discovery in Human Plasma

Over the past decade,
mass spectrometric performance has greatly
improved in terms of sensitivity, dynamic range, and speed. By contrast,
only limited progress has been accomplished with regard to automation,
throughput, and robustness of the proteomic sample preparation process
upstream of mass spectrometry. The present work delivers an optimized
analysis of human plasma samples in both small preclinical and large
clinical studies, enabled by the development of a highly automated
quantitative proteomic workflow. Several iterative evaluation and
validation steps were performed before process “design freeze”
and development completion. A robotic liquid handling workflow and
platform (including reduction, alkylation, digestion, TMT labeling,
pooling, and purification) were shown to provide better quantitative
trueness and precision than manual operation at the bench. Depletion
of the most abundant human plasma proteins and subsequent buffer exchange
were also developed and integrated. Finally, 96 identical pooled human
plasma samples were prepared in a 96-well plate format, and each sample
was individually subjected to our developed workflow. This test revealed
increased throughput and robustness compared with to-date published
manual or less automated workflows. Our workflow is ready-to-use for
future (pre-) clinical studies. We expect our work to facilitate,
accelerate, and improve clinical proteomic discovery in human blood
plasma.