Ferguson analysis of protein electromigration during single-cell electrophoresis in an open microfluidic device

Tan, Kristine Y.; Herr, Amy E.

doi:10.1039/c9an02553g

Cited by 10 publications

(8 citation statements)

References 45 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In the conventional MIBI-TOF imaging workflow, only a thin surface layer (∼200 nm) of the 4-μm thick fixed tissue slice is typically imaged. , However, in the scIB system, diffusion during lysis and electrophoresis may dilute protein in scIB samples more than in fixed tissue, so we hypothesized imaging of scIBs would require deeper sample imaging. Though lateral diffusion of protein signal in scIBs has been well characterized, the depth distribution of signal in scIB protein bands has only been computationally interrogated . During electrophoresis, the motion of charged molecules in the direction of the electric field is governed by the electrostatic Coulomb force and counteracting viscous drag force .…”

Section: Resultsmentioning

confidence: 99%

Multiplexed Ion Beam Imaging Readout of Single-Cell Immunoblotting

et al. 2021

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Resultsmentioning

confidence: 99%

Multiplexed Ion Beam Imaging Readout of Single-Cell Immunoblotting

et al. 2021

Self Cite

View full text Add to dashboard Cite

show abstract

“…However, in the scIB system, diffusion during lysis and electrophoresis may dilute protein in scIB samples more than in fixed tissue, so we hypothesized imaging of scIBs would require deeper sample imaging. Though lateral diffusion of protein signal in scIBs has been well characterized 28 , the depth distribution of signal in scIB protein bands has only been computationally interrogated 29 . During electrophoresis, the motion of charged molecules in the direction of the electric field is governed by the electrostatic Coulomb force and opposite viscous drag 30 .…”

Section: Resultsmentioning

confidence: 99%

Multiplexed Ion Beam Imaging Readout of Single-Cell Immunoblotting

Lomeli

Bossé

Bendall

et al. 2021

Preprint

Self Cite

View full text Add to dashboard Cite

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 spectra 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 find 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.

show abstract

“…In recent years, the electromigration phenomenon, due to several extensive developments, has emerged with numerous successes in separation processes, in the ways of diagnosis assays, [ 28 ] cancer cell detection methods, [ 262,263 ] and also food‐borne pathogen determination techniques. [ 264 ] In addition, state‐of‐the‐art strategies combined with modern devices have been substituted for conventional protocols in electro‐separation and tracking of proteins, nucleic acids, and other biomoieties.…”

Section: Microfluidic‐based Separation Methodsmentioning

confidence: 99%

Molecular Separation by Using Active and Passive Microfluidic chip Designs: A Comprehensive Review

Ebrahimi,

Icoz,

Didarian

et al. 2023

Adv Materials Inter

View full text Add to dashboard Cite

Separation and identification of molecules and biomolecules such as nucleic acids, proteins, and polysaccharides from complex fluids are known to be important due to unmet needs in various applications. Generally, many different separation techniques, including chromatography, electrophoresis, and magnetophoresis, have been developed to identify the target molecules precisely. However, these techniques are expensive and time consuming. “Lab‐on‐a‐chip” systems with low cost per device, quick analysis capabilities, and minimal sample consumption seem to be ideal candidates for separating particles, cells, blood samples, and molecules. From this perspective, different microfluidic‐based techniques have been extensively developed in the past two decades to separate samples with different origins. In this review, “lab‐on‐a‐chip” methods by passive, active, and hybrid approaches for the separation of biomolecules developed in the past decade are comprehensively discussed. Due to the wide variety in the field, it will be impossible to cover every facet of the subject. Therefore, this review paper covers passive and active methods generally used for biomolecule separation. Then, an investigation of the combined sophisticated methods is highlighted. The spotlight also will be shined on the elegance of separation successes in recent years, and the remainder of the article explores how these permit the development of novel techniques.

show abstract

Ferguson analysis of protein electromigration during single-cell electrophoresis in an open microfluidic device

Abstract: Ferguson analysis of protein electromigration from single-cell lysate in an open microfluidic device to inform optimal assay design.

Cited by 10 publications

References 45 publications

Multiplexed Ion Beam Imaging Readout of Single-Cell Immunoblotting

Multiplexed Ion Beam Imaging Readout of Single-Cell Immunoblotting

Multiplexed Ion Beam Imaging Readout of Single-Cell Immunoblotting

Molecular Separation by Using Active and Passive Microfluidic chip Designs: A Comprehensive Review

Contact Info

Product

Resources

About