Arrayed hydrogels pair whole-cell imaging with single-cell mass spectrometry proteomics
- 1University of California, Berkeley
Abstract
Multimodal single-cell analysis aims to elucidate cellular-level phenotype within heterogeneous cell populations. Advancements in single-cell proteomics (SCP) seek to deepen quantitative depth, coverage, and reproducibility for a robust view of functional cell state. However, broadly accessible multimodal approaches are poised to benefit from sample-preparation advancements upstream of the mass spectrometer. Here, we introduce ProteoParcel, a multimodal SCP platform for indexing upstream widefield single-cell images to downstream label-free, bottom-up proteomics. Parcels are spatially arrayed planar polyacrylamide gels patterned with microwells. Each parcel, containing one microwell and an abutting gel region, is designed to integrate the single-cell imaging and SCP analysis modes. First, for whole-cell imaging, each microwell isolates an intact, individual breast cancer cell (MCF-7). After imaging, cells are subjected to in-microwell chemical cell lysis, electro-injection of whole-cell lysate from the microwell into the abutting gel region, in-gel chemical fixation, and finally in-gel tryptic digestion prior to peptide extraction for SCP. Location-indexed parcels are independently releasable to confer single-cell resolution to downstream mass spectrometry. To ensure SCP-suitable proteome solubilization and trypsin/Lys-C digestion, we optimize cell lysis, electrophoresis, and gel pre-equilibration conditions within the gel. Using ProteoParcel, we identify over 1,400 protein species from single, imaged MCF-7 cells. Scrutiny of the gel preparation conditions confirms that hydrogel-lysate interactions introduce predictable, physicochemically interpretable (cell membrane, hydrophobicity) detection biases, while broad functional-class composition and subcellular compartment coverage are preserved when benchmarked to in-solution digestion. ProteoParcel makes facile same-cell multimodal SCP and live-cell imaging.
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BibTeX
@misc{overton2026arrayed,
title = {{Arrayed hydrogels pair whole-cell imaging with single-cell mass spectrometry proteomics}},
author = {M. Overton and C. Deroy and E. Wang and A. Lennon and J. E. Elias and R. McClure and A. E. Herr},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.04.749469},
url = {https://www.biorxiv.org/content/10.64898/2026.09.04.749469v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Arrayed hydrogels pair whole-cell imaging with single-cell mass spectrometry proteomics
AU - M. Overton
AU - C. Deroy
AU - E. Wang
AU - A. Lennon
AU - J. E. Elias
AU - R. McClure
AU - A. E. Herr
PY - 2026
DA - 2026/09/04
PB - bioRxiv
DO - 10.64898/2026.09.04.749469
UR - https://www.biorxiv.org/content/10.64898/2026.09.04.749469v1
AB - Multimodal single-cell analysis aims to elucidate cellular-level phenotype within heterogeneous cell populations. Advancements in single-cell proteomics (SCP) seek to deepen quantitative depth, coverage, and reproducibility for a robust view of functional cell state. However, broadly accessible multimodal approaches are poised to benefit from sample-preparation advancements upstream of the mass spectrometer. Here, we introduce ProteoParcel, a multimodal SCP platform for indexing upstream widefield single-cell images to downstream label-free, bottom-up proteomics. Parcels are spatially arrayed planar polyacrylamide gels patterned with microwells. Each parcel, containing one microwell and an abutting gel region, is designed to integrate the single-cell imaging and SCP analysis modes. First, for whole-cell imaging, each microwell isolates an intact, individual breast cancer cell (MCF-7). After imaging, cells are subjected to in-microwell chemical cell lysis, electro-injection of whole-cell lysate from the microwell into the abutting gel region, in-gel chemical fixation, and finally in-gel tryptic digestion prior to peptide extraction for SCP. Location-indexed parcels are independently releasable to confer single-cell resolution to downstream mass spectrometry. To ensure SCP-suitable proteome solubilization and trypsin/Lys-C digestion, we optimize cell lysis, electrophoresis, and gel pre-equilibration conditions within the gel. Using ProteoParcel, we identify over 1,400 protein species from single, imaged MCF-7 cells. Scrutiny of the gel preparation conditions confirms that hydrogel-lysate interactions introduce predictable, physicochemically interpretable (cell membrane, hydrophobicity) detection biases, while broad functional-class composition and subcellular compartment coverage are preserved when benchmarked to in-solution digestion. ProteoParcel makes facile same-cell multimodal SCP and live-cell imaging.
ER -