Analysis of 8000 proteins and reduced carry over significantly increase the throughput of single-shot proteomics

Abstract

ABSTRACTIn the field of LC-MS based proteomics, increases in sampling depth and proteome coverage have mainly been accomplished by rapid advances in mass spectrometer technology. The comprehensiveness and quality of data that can be generated do however also depend on the performance provided by nano liquid chromatography (nanoLC) separations. Proper selection of reversed-phase separation columns can be of paramount importance to provide the MS instrument with peptides at the highest possible concentration and separated at the highest possible resolution. As an alternative to traditional packed bed LC column technology that uses beads packed into capillary tubing, we present a novel LC column format based on photolithographic definition and Deep Reactive Ion Etching (DRIE) into silicon wafers. With a next generation pillar array column (μPAC) designed for universal use in bottom-up proteomics, the critical dimensions of the stationary phase support structures have been reduced by a factor of 2 to provide further increases in separation power. To demonstrate the potential for single-shot proteomics workflows, we report on a series of optimization and benchmarking experiments where we combine LC separation on a new generation of μPAC columns using Vanquish Neo UHPLC with fast Orbitrap Tribrid MS data-dependent acquisition (DDA) and High-Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS). In addition to providing superior proteome coverage, robust operation over more than 1 month with a single nanoESI emitter and reduction of the column related sample carry over are additional figures of merit that can help improve proteome research sensitivity, productivity and standardization.