Detection of Isoforms Differing by a Single Charge Unit in Individual Cells

Abstract

To measure protein isoforms in individual mammalian cells, we report single-cell resolution isoelectric focusing (scIEF) and high-selectivity immunoprobing. Microfluidic design and photoactivatable materials establish the tunable pH gradients required by IEF and precisely control the transport and handling of each 17-pL cell lysate during analysis. The scIEF assay resolves protein isoforms with resolution down to a single-charge unit, including both endogenous cytoplasmic and nuclear proteins from individual mammalian cells.

Keywords: immunoassays; isoelectric focusing; lab-on-a-chip; proteomics; single-cell analysis.

Figure 1. Direct measurement of proteins using scIEF

(a) Exploded view rendering of scIEF assay setup. (b) Isometric schematic of the multilayer scIEF microdevice and top view photograph of lid layer with catholyte and anolyte regions with blue and red dye, respectively. (c) scIEF workflow. (d) Time-course of tGFP fluorescence signal position during single-cell lysis and scIEF. Error bars indicate band width (4σ). pH range 4–9, microwell position 4.5 mm from catholyte-side edge of the bottom gel. (e) Inverted grayscale fluorescence micrographs report blotting and subsequent immunoprobing (α-tGFP) from a single cell. pH range 4–7, microwell position 6.75 mm from catholyte-side edge of the bottom gel. Traces in arbitrary fluorescence units (AFU).

Figure 2. Control of diffusive and electrokinetic transport to establish robust, non-uniform chemistries for scIEF

(a) Concentration heat maps from simulation show protein diffusion out of the bottom layer is mitigated in hindered (with lid) vs unhindered (no lid, free solution) conditions. Plots indicate maximum concentration along z-axis. Fraction of total protein in bottom layer is f_bl (b) Fluorescence traces show tGFP transfer from bottom layer to lid layer after 10 min of scIEF (c) Inverted grayscale fluorescence micrograph shows focused pH markers in pH 4–7 gradient. Dashed line is linear fit; error bars, peak widths (4σ).

Figure 3. scIEF with immunoprobing resolves proteoforms in individual mammalian cells

(a) False-color fluorescence micrographs and traces show scIEF detection via immunoprobing of β-TUB and tGFP from individual cells. Microwells are outlined with black circle, located at 0 mm. Arrows indicate protein peaks; plotted black outlines, Gaussian fits for identified peaks. (b) False-color fluorescence micrographs and traces show detection of denatured β-TUB isoforms in 3/8 cells. (c) Median total tGFP probing fluorescence (Area under the curve, AUC) under native “N” and denaturing “D” conditions (n_D=8, n_N=9, p<0.01) (d) Relative isoform fractions (n_β-TUB=3, n_tGFP=8). (e) False-color fluorescence micrographs and traces show detection of lamin A/C from individual cells under denaturing conditions (n_D=9). pH range 4–7, microwell position 6.75 mm from catholyte-side edge of the bottom gel in all separations.