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.

Explore plasma proteomics innovations tackling the dynamic range challenge, revealing biomarkers and advancing personalized medicine with Professor Joshua Coon.

In BriefWe present deep analysis of the human proteome in less than 1 h. The Orbitrap Astral mass spectrometer, alongside advances in sample preparation, method development, and data processing, is ushering in a new era of proteomics where a harmony of throughput, depth, and sensitivity are enable studies of unprecedented impact. We contextualize these results within the landscape of human proteomics. Comparisons with previous instrumentation in interrogating gene knockouts demonstrate the potential of proteomics to elevate biological investigation in years to come.