Multiplexed Ion Beam Imaging Readout of Single-Cell Immunoblotting
Gabriela Lomeli, Marc Bosse1, Sean C Bendall1
1Department of Pathology, Stanford University, Stanford, California 94025, United States.
Insights
We developed single-cell immunoblotting with multiplexed ion beam imaging by time-of-flight (scIB-MIBI-TOF) for enhanced proteoform detection. This novel method allows highly multiplexed mass spectrometry-based readout compatible with single-cell analysis in diseases.
Area of Science:
- Biochemistry
- Proteomics
- Mass Spectrometry
Background:
- Single-cell protein analysis is crucial for understanding disease heterogeneity, but current methods like single-cell immunoblotting (scIB) have limited multiplexing due to fluorescence readouts.
- Multiplexed ion beam imaging by time-of-flight (MIBI-TOF) offers high multiplexing potential using metal-tagged antibodies, overcoming spectral overlap issues inherent in fluorescence methods.
Purpose of the Study:
- To develop and validate a novel method combining single-cell immunoblotting (scIB) with multiplexed ion beam imaging by time-of-flight (MIBI-TOF) for highly multiplexed single-cell proteoform analysis.
- To demonstrate the compatibility and efficacy of mass spectrometry-based readout for scIB, enabling deeper insights into cell-to-cell variation in diseases.
Main Methods:
- Developed scIB-MIBI-TOF by integrating scIB with MIBI-TOF, utilizing a polyacrylamide (PA) gel matrix for protein immobilization and metal-tagged antibodies for detection.
- Characterized protein distribution within the 3D PA gel using fluorescence confocal microscopy to optimize MIBI-TOF imaging parameters, including ion dose strength for varying gel depths.
- Performed layer-by-layer MIBI-TOF imaging of the PA gel matrix to analyze proteoforms at the single-cell level.
Main Results:
- Successfully confirmed antibody-protein binding within the PA gel using indirect fluorescence readout of metal-tagged antibodies.
- Determined optimal MIBI-TOF ion dose strengths required for imaging different depths of the PA gel matrix.
- Detected two isoelectrically separated TurboGFP (tGFP) proteoforms from individual glioblastoma cells by imaging approximately 42% of the PA gel depth, showcasing multiplexed mass spectrometry compatibility with scIB.
Conclusions:
- scIB-MIBI-TOF represents the first successful integration of mass spectrometry imaging with single-cell immunoblotting, significantly advancing multiplexed proteoform analysis.
- This technique overcomes the limitations of fluorescence-based readouts, enabling highly multiplexed detection of specific proteoforms within individual cells.
- The demonstrated compatibility of MIBI-TOF with scIB opens new avenues for studying cell-to-cell variations in complex biological systems and diseases like cancer.
Abstract:
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-μm 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 ∼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.
