PT  - JOURNAL ARTICLE
AU  - Benjamin C. Orsburn
AU  - Yuting Yuan
AU  - Namandjé N. Bumpus
TI  - Single Cell Proteomics Using a Trapped Ion Mobility Time-of-Flight Mass Spectrometer Provides Insight into the Post-translational Modification Landscape of Individual Human Cells
AID  - 10.1101/2022.02.12.480144
DP  - 2022 Jan 01
TA  - bioRxiv
PG  - 2022.02.12.480144
4099  - http://biorxiv.org/content/early/2022/02/12/2022.02.12.480144.short
4100  - http://biorxiv.org/content/early/2022/02/12/2022.02.12.480144.full
AB  - Single cell proteomics is a powerful tool with potential for markedly enhancing understanding of cellular processes. Previously reported single cell proteomics innovations employ Orbitrap mass spectrometers. In this study we describe the development, optimization, and application of multiplexed single cell proteomics to the analysis of human-derived cells using trapped ion mobility time-of-flight mass spectrometry. This method, denoted as pasefRiQ is an advance as it allows accurate peptide quantification as demonstrated by a two-proteome standard model, with little variation in accuracy in samples at picogram peptide concentrations. When employing a peptide carrier channel to boost protein sequence coverage, we obtain over 40,000 tandem mass spectra in 30 minutes, providing unprecedented sequence coverage of each identified protein. Using NCI-H-358 cells, which are a human bronchioalveolar carcinoma and KRASG12C model cell line, we demonstrate that the level of coverage achieved using this method enables the quantification of up to 1,255 proteins per cell and the detection of multiple classes of post-translational modifications in single cells. Further, when the cells were treated with AMG510, a KRASG12C covalent inhibitor, pasefRiQ revealed cell-to-cell variability in the impact of the drug on the NCI-H-358 proteome. In fact, when examining proteins that changed in protein abundance in response to AMG510 we can identify those that are disproportionately increased or decreased in a small number of cells relative to the total population of cells that were cultured and treated simultaneously. These results suggest single cell proteomics through pasefRiQ can provide powerful insight for cellular and drug mechanism studies.Summary This work describes the establishment of a single cell proteomics method using a time-of-flight mass spectrometer. Through this approach, we demonstrate the confident identification of post-translational modifications in single human-derived cells. Additionally, using a KRASG12C covalent inhibitor as a model compound we show that this method can be used to understand pharmacological responses of single human-derived cultured cells.Competing Interest StatementThe authors have declared no competing interest.