期刊
ANALYTICAL CHEMISTRY
卷 93, 期 24, 页码 8517-8525出版社
AMER CHEMICAL SOC
DOI: 10.1021/acs.analchem.1c01050
关键词
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资金
- National Institutes of Health [R01CA203018]
- National Cancer Institute of the National Institutes of Health, Cancer Moonshot award [1R33CA225296-01]
- Chan Zuckerberg Biohub
- National Science Foundation Graduate Research Fellowship Program (NSF GRFP)
- Gordon and Betty Moore Foundation
- Bill and Melinda Gates Foundation
- [1R01AG056287]
- [1R01AG057915]
- [1U24CA224309]
- [1-DP5-OD019822]
Improvements in single-cell protein analysis are necessary for studying the cell-to-cell variation seen in diseases like cancer. MIBI-TOF is a mass spectrometry imaging technology that allows for highly multiplexed protein detection at the single-cell level.
Improvements in single-cell protein analysis are required to study the cell-to-cell variation inherent to diseases, including cancer. Single-cell immunoblotting (scIB) offers proteoform detection specificity, but often relies on fluorescence-based readout and is therefore limited in multiplexing capability. Among rising multiplexed imaging methods is multiplexed ion beam imaging by time-of-flight (MIBI-TOF), a mass spectrometry imaging technology. MIBI-TOF employs metal-tagged antibodies that do not suffer from spectral overlap to the same degree as fluorophore-tagged antibodies. We report for the first-time MIBI-TOF of single-cell immunoblotting (scIB-MIBI-TOF). The scIB assay subjects single-cell lysate to protein immunoblotting on a microscale device consisting of a 50- to 75-mu m thick hydrated polyacrylamide (PA) gel matrix for protein immobilization prior to in-gel immunoprobing. We confirm antibody-protein binding in the PA gel with indirect fluorescence readout of metal-tagged antibodies. Since MIBI-TOF is a layer-by-layer imaging technique, and our protein target is immobilized within a 3D PA gel layer, we characterize the protein distribution throughout the PA gel depth by fluorescence confocal microscopy and confirm that the highest signal-to-noise ratio is achieved by imaging the entirety of the PA gel depth. Accordingly, we report the required MIBI-TOF ion dose strength needed to image varying PA gel depths. Lastly, by imaging similar to 42% of PA gel depth with MIBI-TOF, we detect two isoelectrically separated TurboGFP (tGFP) proteoforms from individual glioblastoma cells, demonstrating that highly multiplexed mass spectrometry-based readout is compatible with scIB.
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