Gel-Confined Rolling-Circle Amplification Enables Sensitive Single-Cell Proteoform Analysis

Abstract

Protein abundance alone does not capture the molecular diversity generated by protein processing and post-translational modification, yet most single-cell protein assays do not resolve these proteoform states. Here, we develop RCAmp-scWB, a post-separation amplification strategy that performs rolling-circle amplification directly within the polyacrylamide gel after single-cell protein electrophoresis. Reaction-transport modeling identifies a balance between reagent access and confinement of the template-associated amplification product that supports signal amplification while retaining the electrophoretically encoded spatial readout. Using purified protein standards, RCAmp-scWB produced a 4.0-7.8-fold steeper concentration-response slope than conventional single-cell western blotting. Across six human cancer cell lines, RCAmp-scWB quantified protein abundance and resolved distinct Vimentin and PD-L1 proteoform states whose relative abundance and single-cell distributions were not captured by total protein measurements alone. Extension to individual small extracellular vesicles further revealed source-dependent shifts in Vimentin proteoform composition despite comparatively similar abundance of one major Vimentin species. By separating electrophoretic molecular discrimination from signal amplification, RCAmp-scWB enables proteoform-resolved analysis of heterogeneous cells and small extracellular vesicles.

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BibTeX RIS

BibTeX

@misc{zhao2026gelconfined,
  title = {{Gel-Confined Rolling-Circle Amplification Enables Sensitive Single-Cell Proteoform Analysis}},
  author = {Luyao Zhao and Yanjun Yang and Yaochao Zheng and Yuhao Zhang and Zhengfu Huang and William Teng and Lucas Yatcyshyn and Jiahwei Cheong and Jonathan Arnold and Jin Xie and Kenan Song and Xianqiao Wang and Yiping Zhao and Xianyan Chen and Yao Yao and Leidong Mao and Yang Liu},
  howpublished = {arXiv},
  year = {2026},
  month = sep,
  doi = {10.48550/arxiv.2609.36251},
  eprint = {2609.36251},
  archivePrefix = {arXiv},
  url = {https://arxiv.org/abs/2609.36251},
}

RIS

TY  - UNPB
TI  - Gel-Confined Rolling-Circle Amplification Enables Sensitive Single-Cell Proteoform Analysis
AU  - Luyao Zhao
AU  - Yanjun Yang
AU  - Yaochao Zheng
AU  - Yuhao Zhang
AU  - Zhengfu Huang
AU  - William Teng
AU  - Lucas Yatcyshyn
AU  - Jiahwei Cheong
AU  - Jonathan Arnold
AU  - Jin Xie
AU  - Kenan Song
AU  - Xianqiao Wang
AU  - Yiping Zhao
AU  - Xianyan Chen
AU  - Yao Yao
AU  - Leidong Mao
AU  - Yang Liu
PY  - 2026
DA  - 2026/09/28
PB  - arXiv
DO  - 10.48550/arxiv.2609.36251
UR  - https://arxiv.org/abs/2609.36251
AB  - Protein abundance alone does not capture the molecular diversity generated by protein processing and post-translational modification, yet most single-cell protein assays do not resolve these proteoform states. Here, we develop RCAmp-scWB, a post-separation amplification strategy that performs rolling-circle amplification directly within the polyacrylamide gel after single-cell protein electrophoresis. Reaction-transport modeling identifies a balance between reagent access and confinement of the template-associated amplification product that supports signal amplification while retaining the electrophoretically encoded spatial readout. Using purified protein standards, RCAmp-scWB produced a 4.0-7.8-fold steeper concentration-response slope than conventional single-cell western blotting. Across six human cancer cell lines, RCAmp-scWB quantified protein abundance and resolved distinct Vimentin and PD-L1 proteoform states whose relative abundance and single-cell distributions were not captured by total protein measurements alone. Extension to individual small extracellular vesicles further revealed source-dependent shifts in Vimentin proteoform composition despite comparatively similar abundance of one major Vimentin species. By separating electrophoretic molecular discrimination from signal amplification, RCAmp-scWB enables proteoform-resolved analysis of heterogeneous cells and small extracellular vesicles.
ER  -

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