Abstract
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 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, while achieving higher sequence coverage of each identified protein than described for SCOPE2. 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 cells were treated with sotorasib, a KRASG12C covalent inhibitor, pasefRiQ revealed cell-to-cell variability in the impact of the drug on the NCI-H-358 cells, providing insight missed by traditional analyses. We provide multiple resources necessary for the application of single cell proteomics to drug treatment studies including tools to reduce cell cycle linked proteomic effects from masking pharmacological phenotypes.
Significance Statement 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 Statement
The authors have declared no competing interest.
Footnotes
Competing Interest Statement: We have no competing interests to disclose.
This version of the manuscript contains multiple changes made to address peer reviewer comments and concerns. Highlights include further analysis into the "carrier proteome effect" observed when using this method, with particular focus on the range between 10x and 500x carrier that has been the source of recent controversy on Orbitrap D20 instrumentation. In addition, the creation of new tools allow the direct extraction of cell cycle effects from our drug treatment data. We further investigate the mechanism of sotorasib treatment on single H358 cells by integrating single cell RNAseq results into this analysis. Through a more thorough data analysis pipeline and exhaustive manual interpretation of our data we provide evidence of 7 classes of protein post-translational modifications that can be quantified on high abundance proteins in single human cells.