Deep proteome profiling with reduced carry over using superficially porous microfabricated nanoLC columns

Abstract

In 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.

Wide Window Acquisition and AI-based data analysis to reach deep proteome coverage for a wide sample range, including single cell proteomic inputs

“…To our surprise, a striking protein ID gap between an injection amount of 10 ng and 50 ng was observed, which had not been reported by earlier studies in our lab. 10 In addition, increasing the peptide load led to a less pronounced increase in protein IDs. Only for longer gradients, the protein ID gap between different peptide loads showed a wider spread.…”

“…This enables fast loading, washing and conditioning of the column pre-and post-analysis increasing sample throughput. 10,11 This is combined with a wide window acquisition (WWA) DDA method, which merges the strengths of DDA and DIA. As indicated by the name, we use a broad isolation window of ≥4 m/z in an otherwise data dependent precursor selection.…”

“…9 Furthermore, the use of superficially porous material on the surface of these micropillars instead of classical fully porous beads reduces the persistent adsorption of hydrophobic peptides, thereby reducing carry-over. 10 By using rectangular shaped pillars, typical flow path lengths around 50 cm can be realized in very short physical column lengths of only 5.5 cm resulting in low backpressure at nano flow rates and a broad dynamic range of available flow rates ≤2.5µL/min. This enables fast loading, washing and conditioning of the column pre- and post-analysis increasing sample throughput.…”

“…This enables fast loading, washing and conditioning of the column pre- and post-analysis increasing sample throughput. 10,11…”

Segmented Microfluidics-Based Packing Technology for Chromatographic Columns

“…The recently reported proteomics applications of such columns demonstrated superb kinetics, which microfluidic columns cannot compare with at the current stage. 52,53 In practical terms, however, the limited loadability of the pillar array and open tubular columns cannot compete with particle-packed columns at this moment. In this sense, the microfluidic packed meter-long columns have a practical transferability to proteomics laboratories although more analytical time is needed to realize deep-mining proteomics.…”