Multiplexed Ion Beam Imaging Readout of Single-Cell Immunoblotting

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.

Multiplex Protein Profiling by Low-Signal-Loss Single-Cell Western Blotting with Fluorescent-Quenching Aptamers

“…35 Second, in these studies, a 2 h duration for antibody incubation has been widely adopted and recognized as a standard procedure. 5,8,36 Therefore, we followed this well-established practice to ensure consistency and comparability with the existing scWB protocols.…”

“…In addition, the large molecular weight of the antibody (typically 150−1000 kDa) limits its penetration and diffusion ability in the hydrogel. 8,9 Typically, high concentrations of antibodies (2.5−5 μg/μL) are employed to ensure the binding of antigens and antibodies. 5,8 Also, in scWB, potential signal overlap exists between protein bands, resulting in interference among fluorescent signals.…”

“…8,9 Typically, high concentrations of antibodies (2.5−5 μg/μL) are employed to ensure the binding of antigens and antibodies. 5,8 Also, in scWB, potential signal overlap exists between protein bands, resulting in interference among fluorescent signals. Nevertheless, the detectable range of protein molecular weights, as determined by the concentration of acrylamide, is also limited by the usage of antibodies.…”

The Intriguing Landscape of Single‐Cell Protein Analysis

“…More recently, Lomeli et al introduced scWB with a MIBI-TOF readout by using a metal-tagged secondary antibody, endowed the scWB method with multiplexing capability. [79] Moreover, antibody incubation is one of the most critical steps in the in-gel immunoblot-ting method, as the entry of antibody molecules into a wetted hydrogel is hindered by size-exclusion, hydrophobic-hydrophobic, and electrostatic interactions. In this respect, many efforts have been made to increase the concentration of antibodies in the gels, which largely improves the antibody incubation performance in the scWB.…”

“…Despite recent developments in single-cell proteomic technologies, challenges remain in getting a deep understanding of the complex and dynamic nature of proteome in single cells. For example, there are many complex biological factors taking place including splice variants, PTMs, large concentration dy- Single-cell barcode chip (SCBC) Single-cell lysis Fluorescence detection About 40 Medium High [14][15][16][17][18][19][20][21][22][23] Miniaturized ELISA Single-cell Fluorescence detection About 30 Medium Medium [37][38][39] Droplet-based microfluidics methods Single-cell Fluorescence detection About 10 Medium Medium [40][41][42][43][44][45][46] CE-LIF Single-cell Fluorescence detection About 10 Medium Medium [ 47,48] Single-cell Western blotting (scWB) Single-cell lysis Fluorescence detection About 10 Medium Medium [72][73][74][75][76][77][78][79][80][81] Proximity ligation assay-based method Single-cell RNA sequencing Unlimited Medium High [87][88][89][90][91][92][93][94][95][96]…”