Ultrasensitive NanoLC-MS of Subnanogram Protein Samples Using Second Generation Micropillar Array LC Technology with Orbitrap Exploris 480 and FAIMS PRO

[Image: see text] In the light of the ongoing single-cell revolution, scientific disciplines are combining forces to retrieve as much relevant data as possible from trace amounts of biological material. For single-cell proteomics, this implies optimizing the entire workflow from initial cell isolation down to sample preparation, liquid chromatography (LC) separation, mass spectrometer (MS) data acquisition, and data analysis. To demonstrate the potential for single-cell and limited sample proteomics, we report on a series of benchmarking experiments where we combine LC separation on a new generation of micropillar array columns with state-of-the-art Orbitrap MS/MS detection and high-field asymmetric waveform ion mobility spectrometry (FAIMS). This dedicated limited sample column has a reduced cross section and micropillar dimensions that have been further downscaled (interpillar distance and pillar diameter by a factor of 2), resulting in improved chromatography at reduced void times. A dilution series of a HeLa tryptic digest (5–0.05 ng/μL) was used to explore the sensitivity that can be achieved. Comparative processing of the MS/MS data with Sequest HT, MS Amanda, Mascot, and SpectroMine pointed out the benefits of using Sequest HT together with INFERYS when analyzing sample amounts below 1 ng. Here, 2855 protein groups were identified from just 1 ng of HeLa tryptic digest hereby increasing detection sensitivity as compared to a previous contribution by a factor well above 10. By successfully identifying 1486 protein groups from as little as 250 pg of HeLa tryptic digest, we demonstrate outstanding sensitivity with great promise for use in limited sample proteomics workflows.