Dataset Information

Proteomic Analysis of Single Mammalian Cells Enabled by Microfluidic Nanodroplet Sample Preparation and Ultrasensitive NanoLC-MS.

ABSTRACT: We report on the quantitative proteomic analysis of single mammalian cells. Fluorescence-activated cell sorting was employed to deposit cells into a newly developed nanodroplet sample processing chip, after which samples were analyzed by ultrasensitive nanoLC-MS. An average of circa 670 protein groups were confidently identified from single HeLa cells, which is a far greater level of proteome coverage for single cells than has been previously reported. We demonstrate that the single-cell proteomics platform can be used to differentiate cell types from enzyme-dissociated human lung primary cells and identify specific protein markers for epithelial and mesenchymal cells.

SUBMITTER: Zhu Y

PROVIDER: S-EPMC6261339 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Proteomic Analysis of Single Mammalian Cells Enabled by Microfluidic Nanodroplet Sample Preparation and Ultrasensitive NanoLC-MS.

Zhu Ying Y Clair Geremy G Chrisler William B WB Shen Yufeng Y Zhao Rui R Shukla Anil K AK Moore Ronald J RJ Misra Ravi S RS Pryhuber Gloria S GS Smith Richard D RD Ansong Charles C Kelly Ryan T RT

Angewandte Chemie (International ed. in English) 20180614 38

We report on the quantitative proteomic analysis of single mammalian cells. Fluorescence-activated cell sorting was employed to deposit cells into a newly developed nanodroplet sample processing chip, after which samples were analyzed by ultrasensitive nanoLC-MS. An average of circa 670 protein groups were confidently identified from single HeLa cells, which is a far greater level of proteome coverage for single cells than has been previously reported. We demonstrate that the single-cell proteom ...[more]

PMID: 29797682

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Project description:Immunosubtraction is a powerful and resource-intensive laboratory medicine assay that reports both protein mobility and binding specificity. To expedite and automate this electrophoretic assay, we report on advances to the electrophoretic immunosubtraction assay by introducing a homogeneous, not heterogeneous, format with integrated sample preparation. To accomplish homogeneous immunosubtraction, a step-decrease in separation matrix pore-size at the head of a polyacrylamide gel electrophoresis (PAGE) separation channel enables "subtraction" of target analyte when capture antibody is present (as the large immune-complex is excluded from PAGE), but no subtraction when capture antibody is absent. Inclusion of sample preparation functionality via small pore size polyacrylamide membranes is also key to automated operation (i.e., sample enrichment, fluorescence sample labeling, and mixing of sample with free capture antibody). Homogeneous sample preparation and assay operation allows on-the-fly, integrated subtraction of one to multiple protein targets and reuse of each device. Optimization of the assay is detailed which allowed for ~95% subtraction of target with 20% non-specific extraction of large species at the optimal antibody-antigen ratio, providing conditions needed for selective target identification. We demonstrate the assay on putative markers of injury and inflammation in cerebrospinal fluid (CSF), an emerging area of diagnostics research, by rapidly reporting protein mobility and binding specificity within the sample matrix. We simultaneously detect S100B and C-reactive protein, suspected biomarkers for traumatic brain injury (TBI), in ~2 min. Lastly, we demonstrate S100B detection (65 nM) in raw human CSF with an estimated lower limit of detection of 3.25 nM, within the clinically relevant concentration range for detecting TBI in CSF. Beyond the novel CSF assay introduced here, a fully automated immunosubtraction assay would impact a spectrum of routine but labor and time-intensive laboratory medicine assays.

Dataset Information

Proteomic Analysis of Single Mammalian Cells Enabled by Microfluidic Nanodroplet Sample Preparation and Ultrasensitive NanoLC-MS.

Publications

Proteomic Analysis of Single Mammalian Cells Enabled by Microfluidic Nanodroplet Sample Preparation and Ultrasensitive NanoLC-MS.

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