Abstract
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.
Highlights
Single-cell proteomics can reveal cellular phenotypic heterogeneity and cell-specific functional networks underlying biological processes
We showed that dataindependent acquisition (DIA) mass spectrometry (MS), which detects all precursors and fragments in the entire m/z range within isolation windows, enabled all retrospective peptide mapping against spectral libraries and offered 2.3-fold superior coverage than conventional datadependent acquisition (DDA) mode[28]
To provide a streamlined microproteomic pipeline for mass-limited samples, we designed a microfluidic device as an integrated proteomics chip to offer all-in-one functionality from cell input to complete proteomic sample processing
Summary
Single-cell proteomics can reveal cellular phenotypic heterogeneity and cell-specific functional networks underlying biological processes. Incorporation of isotopic labeling to extend the multiplexity ability was demonstrated at the level of 1000 proteins in primary cells[12,24] With these advances, a fully automated workflow, starting from multiplexed cell capturing and imaging, cell lysis, and protein digestion to peptide desalting, all integrated within a single device to realize proteomic analysis for low-input samples has not yet been established, despite the prospect to substantially minimize sample loss and achieve high reproducibility and sensitivity. An integrated proteomics chip (iProChip, 1–100 cells) and its extended version for single-cell capacity (SciProChip) were designed and coupled with data-independent acquisition (DIA) MS as streamlined nanoproteomics (nanogram of cells) pipelines These chips are designed as automated stations for the entire proteomic workflow, offering built-in features including quantifiable cell capture and imaging, complete cell lysis, protein digestion, and peptide desalting. The presented workflow illustrates a unique implementation of microfluidic devices with all-in-one functionality to achieve automated and streamlined proteomic preparation, which offers high sensitivity and reproducibility for limited input samples, including a single cell
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