SynchroSep-MS: Parallel LC Separations for Multiplexed Proteomics

Achieving high throughput remains a challenge in MS-based
proteomics
for large-scale applications. We introduce SynchroSep-MS, a novel
method for parallelized, label-free proteome analysis that leverages
the rapid acquisition speed of modern mass spectrometers. This approach
employs multiple liquid chromatography columns, each with an independent
sample, simultaneously introduced into a single mass spectrometer
inlet. A precisely controlled retention time offset between sample
injections creates distinct elution profiles, facilitating unambiguous
analyte assignment. We modified the DIA-NN workflow to effectively
process these unique parallelized data, accounting for retention time
offsets. Using a dual-column setup with mouse brain peptides, SynchroSep-MS
detected approximately 16,700 unique protein groups, nearly doubling
the peptide information obtained from a conventional single proteome
analysis. The method demonstrated excellent precision and reproducibility
(median protein %RSDs less than 4%) and high quantitative linearity
(median R2 greater than 0.96) with minimal matrix interference.
SynchroSep-MS represents a new paradigm for data collection and the
first example of label-free multiplexed proteome analysis via parallel
LC separations, offering a direct strategy to accelerate throughput
for demanding applications such as large-scale clinical cohorts and
single-cell analyses without compromising peak capacity or causing
ionization suppression.