Streamlined single-cell proteomics by an integrated microfluidic chip and data-independent acquisition mass spectrometry

• Published in: Nature communications Vol. 13; no. 1; pp. 37 - 13
• Main Authors: Gebreyesus, Sofani Tafesse, Siyal, Asad Ali, Kitata, Reta Birhanu, Chen, Eric Sheng-Wen, Enkhbayar, Bayarmaa, Angata, Takashi, Lin, Kuo-I, Chen, Yu-Ju, Tu, Hsiung-Lin
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 10.01.2022; Nature Publishing Group; Nature Portfolio
• Subjects: 631/1647/277; 631/1647/296; 631/45/612/1248; 631/61/475; 82/58; 82/62; Animals; Biological activity; Cell Line, Tumor; Cell Separation; Cellular communication; Heterogeneity; Humanities and Social Sciences; Humans; Lab-On-A-Chip Devices; Lung Neoplasms; Mammalian cells; Mass spectrometry; Mass Spectrometry - methods; Mass spectroscopy; Microfluidics; Microfluidics - methods; multidisciplinary; Proteome; Proteomes; Proteomics; Proteomics - methods; Reproducibility of Results; Sample preparation; Science; Science (multidisciplinary); Scientific imaging; Spectroscopy; Workflow
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-021-27778-4

Single-cell proteomics can reveal cellular phenotypic heterogeneity and cell-specific functional networks underlying biological processes. Here, we present a streamlined workflow combining microfluidic chips for all-in-one proteomic sample preparation and data-independent acquisition (DIA) mass spectrometry (MS) for proteomic analysis down to the single-cell level. The proteomics chips enable multiplexed and automated cell isolation/counting/imaging and sample processing in a single device. Combining chip-based sample handling with DIA-MS using project-specific mass spectral libraries, we profile on average ~1,500 protein groups across 20 single mammalian cells. Applying the chip-DIA workflow to profile the proteomes of adherent and non-adherent malignant cells, we cover a dynamic range of 5 orders of magnitude with good reproducibility and <16% missing values between runs. Taken together, the chip-DIA workflow offers all-in-one cell characterization, analytical sensitivity and robustness, and the option to add additional functionalities in the future, thus providing a basis for advanced single-cell proteomics applications. Single-cell proteomics is an emerging approach to characterize cell-to-cell differences. Here, the authors develop chips that enable complete proteomic sample processing down to the single-cell level and integrate them with DIA-MS into a streamlined single-cell proteomics workflow.