High-Throughput Proteomics Enabled by a Fully Automated Dual-Trap and Dual-Column LC-MS

This Technical Note describes a dual-column liquid chromatography
system coupled to mass spectrometry (LC-MS) for high-throughput bottom-up
proteomic analysis. This system made full use of two 2-position 10-port
valves and a binary pump with an integrated loading pump of a commercial
LC instrument to provide successive operation of two parallel subsystems.
Each subsystem consisted of a set of trap columns and an analytical
column. A T-junction union was used to split the mobile phase from
the loading pump into two parts. This allowed one set of columns to
be washed and equilibrated, followed by the injection of the next
sample, while the previous sample was eluting and being analyzed on
the other set of columns, thereby greatly increasing the analysis
throughput. This approach showed high reproducibility for the analysis
of HeLa tryptic digests with average relative standard deviation
(RSD) values of 1.75%, 6.90%, and 5.19% for the identification number
of proteins, peptides, and peptide-spectrum matches (PSMs), respectively,
across 10 consecutive runs. The capacity for peptide and protein identification,
as well as proteome depth, of the dual-column LC system was comparable
to a conventional single-column system. Due to its simple equipment
requirements and set up process, this method should be highly accessible
for other laboratories.