Dual LC–MS Platform for High-Throughput Proteome Analysis

We describe a dual-column
interface for parallel chromatography
to improve throughput during LC–MS experimentation. The system
employs a high-voltage switch to operate two capillary column/nanospray
emitters fixed at the MS orifice. Sequentially loading one column
while operating the second nearly doubles the LC–MS duty cycle.
Given the innate run-to-run variation of a nanospray LC–MS
(12% RSD peak area; 2% retention time), the intercolumn variability
of the platform showed no meaningful difference for proteome analysis,
with equal numbers of proteins and peptides identified per column.
Applied to GeLC analysis of an E. coli extract, throughput
was increased using one of three methods: doubling the number of replicates,
increasing the LC gradient length, or sectioning the gel into twice
as many fractions. Each method increased the total number of identifications
as well as detection throughput (number of peptides/proteins identified
per hour). The greatest improvement was achieved by doubling the number
of gel slices (10 vs 5). Analysis on the dual column platform provided
a 26% increase in peptides identified per hour (24% proteins). This
translates into ∼50% more total proteins and peptides identified
in the experiment using the dual LC–MS platform.