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bioRxiv · 10.1101/2025.06.23.660827

Evaluation of the nano Liquid Chromatography coupled with Zeno TOF 7600 Mass Spectrometer for Cell Type-Resolved Spatial Proteomics

Zhang, K.·Wang, X.·He, W.·Wang, J.·He, A.·Zheng, B.·Li, Y.·Xu, R.·Gao, W.·Lam, H. H. N.·Tian, R.
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Abstract

In this study, we systematically evaluated the nanoLC-Zeno TOF platform using data-independent acquisition (DIA) and identified 3,142 to 7,059 protein groups from 1 to 200 ng of standard K562 digest. Additionally, a targeted acquisition strategy detected 184 peptides with enhanced signal using Zeno activation. To achieve spatial cell-type resolved proteomics, we developed a workflow integrating image-guided laser capture microdissection for analysis of gastric cancer tissue. A 30-minute nanoLC gradient facilitated the quantification of 2,514 proteins from 0.2 mm{superscript 2} regions representing cancer cells, cancer-associated fibroblasts (CAFs), and immune cells, as defined by multiplex immunohistochemistry. This spatial proteomics approach revealed cellular heterogeneity within the tumor microenvironment of a clinical gastric cancer sample, and targeted validation identified differential expression of 12 cancer-associated proteins comprising 103 peptides between cancer cell-enriched and CAFs-enriched regions. This workflow offers a practical strategy for spatial visual proteomic analysis in clinical tissue samples.

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Zhang, K., Wang, X., He, W., Wang, J., He, A., Zheng, B., Li, Y., Xu, R., Gao, W., Lam, H. H. N., Tian, R.. 2025-06-27. Evaluation of the nano Liquid Chromatography coupled with Zeno TOF 7600 Mass Spectrometer for Cell Type-Resolved Spatial Proteomics. https://doi.org/10.1101/2025.06.23.660827

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