From genomics to transcriptomics to proteomics, microfluidic tools underpin recent advances in single-cell biology. Detection of specific proteoforms-with single-cell resolution-presents challenges in detection specificity and sensitivity. Miniaturization of protein immunoblots to single-cell resolution mitigates these challenges. For example, in microfluidic western blotting, protein targets are separated by electrophoresis and subsequently detected using fluorescently labeled antibody probes. To quantify the expression level of each protein target, the fluorescent protein bands are fit to Gaussians; yet, this method is difficult to use with noisy, low-abundance, or low-SNR protein bands, and with significant band skew or dispersion. In this study, we investigate segmentation-based approaches to robustly quantify protein bands from single-cell protein immunoblots. As compared to a Gaussian fitting pipeline, the segmentation pipeline detects >1.5× more protein bands for downstream quantification as well as more of the low-abundance protein bands (i.e., with SNR ∼3). Utilizing deep learning-based segmentation approaches increases the recovery of low-SNR protein bands by an additional 50%. However, we find that segmentation-based approaches are less robust at quantifying poorly resolved protein bands (separation resolution, Rs < 0.6). With burgeoning needs for more single-cell protein analysis tools, we see microfluidic separations as benefitting substantially from segmentation-based analysis approaches.
Keywords: Immunoassay; Proteoform; Single cell; Western blotting.
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