The analysis of ultra-low input samples or even individual cells is essential to answering a multitude of biomedical questions, but current proteomic workflows are limited in their sensitivity and reproducibility. Here we report a comprehensive workflow that includes optimized strategies for all steps from cell lysis to data analysis. Thanks to convenient to handle 1 uL sample volume and standardized 384 well plates the workflow is easy for even novice users to implement. At the same time, it can be performed semi-automatized using the CellenONE, which allows for highest reproducibility. To achieve high throughput, ultrashort gradient lengths down to 5 min were tested using advanced u-pillar columns. Data-dependent acquisition (DDA), wide-window acquisition (WWA) and data-independent acquisition (DIA), and commonly used advanced data-analysis algorithms were bench-marked. Using DDA, 1790 proteins covering a dynamic range of four orders of magnitude were identified in a single cell. Using DIA, proteome coverage increased to more than 2200 proteins identified from single cell level input in a 20-min active gradient. The workflow enabled differentiation of two cell lines, demonstrating its suitability to cellular heterogeneity determination.
[doi:10.25345/C5XW4858S]
[dataset license: CC0 1.0 Universal (CC0 1.0)]
Keywords: Single Cell Proteomics ; one-pot workflow ; label-free ; WWA ; DIA ; uPAC ; CellenONE
Principal Investigators: (in alphabetical order) |
Karl Mechtler, IMP, IMBA, N/A Manuel Matzinger, IMP, Austria |
Submitting User: | manuel_matzinger1 |
Matzinger M, Müller E, Dürnberger G, Pichler P, Mechtler K.
Robust and Easy-to-Use One-Pot Workflow for Label-Free Single-Cell Proteomics.
Anal Chem. 2023 Mar 7;95(9):4435-4445. Epub 2023 Feb 20.
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