A Universal and High-Throughput Proteomics Sample Preparation Platform

Major
advances have been made to improve the sensitivity of mass
analyzers, spectral quality, and speed of data processing enabling
more comprehensive proteome discovery and quantitation. While focus
has recently begun shifting toward robust proteomics sample preparation
efforts, a high-throughput proteomics sample preparation is still
lacking. We report the development of a highly automated universal
384-well plate sample preparation platform with high reproducibility
and adaptability for extraction of proteins from cells within a culture
plate. Digestion efficiency was excellent in comparison to a commercial
digest peptide standard with minimal sample loss while improving sample
preparation throughput by 20- to 40-fold (the entire process from
plated cells to clean peptides is complete in ∼300 min). Analysis
of six human cell types, including two primary cell samples, identified
and quantified ∼4,000 proteins for each sample in a single
high-performance liquid chromatography (HPLC)–tandem mass spectrometry
injection with only 100–10K cells, thus demonstrating universality
of the platform. The selected protein was further quantified using
a developed HPLC-multiple reaction monitoring method for HeLa digests
with two heavy labeled internal standard peptides spiked in. Excellent
linearity was achieved across different cell numbers indicating a
potential for target protein quantitation in clinical research.