High-flow Nano-chromatography Columns Facilitate Rapid and High-quality Proteome Analysis on Standard Nano-LC Hardware

Abstract

Optimizing the acquisition of proteomics data collected from a mass spectrometer (MS) requires careful selection of processed material quantities, liquid-chromatography (LC) setup, and data acquisition parameters. The small internal diameter (ID) columns standardly used in nano-chromatography coupled MS result in long per injection overhead times that require sacrifices in design of offline-fractionation and data acquisition schemes. As cohort sizes and the numbers of samples to be analyzed continue to increase, there is a need to investigate methods for improving the efficiency and time of an acquisition (LC + MS). In this work, the ability to improve throughput in single runs or as part of an in-depth proteome analysis of a fractionated sample using standard LC hardware is investigated. Capitalizing on the increased loading capacity of nanochromatography columns with larger IDs, substantially improved throughput with no reduction in detection sensitivity is achieved in single-injection proteome analyses. An optimized 150 m ID column setup is paired with an offline fractionation-concatenation scheme to demonstrate the ability to perform in-depth proteome analysis on-par with current state-of-the-art studies, while minimizing sample loading overhead. Together, these data demonstrate an easy and effective means to improve sample analysis throughput with no reduction in data quality using an approach that is applicable to any standard nano-LC hardware.