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

more »

... a series of benchmarking experiments where we combine LC separation on a new generation of micro pillar 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 micro pillar dimensions that have been further downscaled (inter pillar 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 per microliter) 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 samples amounts below 1 ng. 2855 proteins were identified from just 1 ng of HeLa lysate, hereby increasing detection sensitivity as compared to a previous contribution by a factor well above 10. By successfully identifying 1486 proteins from as little as 250 pg of HeLa tryptic digest, we demonstrate outstanding sensitivity with great promise for use in limited sample proteomics workflows.