Abstract
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.
Highlights
During the past few years, the sensitivity of liquid chromatography (LC)-mass spectrometer (MS)/MS instrumentation has evolved to a level where consistent identification and quantification of proteins from single cells has become feasible.[1−5] As opposed to LC-MS/MS analysis of proteins originating from bulk cell populations, proteome analysis of a single or a few selected cells allows attributing biological characteristics to individual cells
LC-MS/MS analyses performed at an interval of 4 h clearly indicate the positive effect on peptide abundance when using 0.001% PEG as an additive into the sample injection solution (Figure S2)
Several research groups have demonstrated the potential of using ultra low flow LC as an effective approach to increase detection sensitivity in LC-MS based proteomics
Summary
During the past few years, the sensitivity of LC-MS/MS instrumentation has evolved to a level where consistent identification and quantification of proteins from single cells has become feasible.[1−5] As opposed to LC-MS/MS analysis of proteins originating from bulk cell populations, proteome analysis of a single or a few selected cells allows attributing biological characteristics to individual cells. When injecting 1 ng of HeLa digest, we found that working at a CV of −50 V produces on average 44% more protein group identifications compared to the same analysis without FAIMS These findings are consistent with earlier reports and confirm that single CV FAIMS reduces the sampling of highly abundant proteins, resulting in higher proteome coverage but with lower peptide sequence coverage per protein.[28] To investigate the potential of using methods with internal CV stepping for the analysis of low sample amounts, a series of exploratory experiments was set up where multiple CVs were used within a single analysis. This is expected and can be attributed to the fact that it is less likely for low abundant peptides to generate clean MS chromatograms that allow proper quantification
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