Evaluation of the nano Liquid Chromatography coupled with Zeno TOF 7600 Mass Spectrometer for Cell Type-Resolved
Keren Zhang1, Xi Wang2, Wan He3
1Department of Oncology, The Second Clinical Medical College, The First Affiliated Hospital, Jinan University (Shenzhen People's Hospital, the First Affiliated Hospital of Southern University of Science and Technology), Shenzhen, 518020, China; Shenzhen Key Laboratory of Functional Proteomics, Research Center for Chemical Biology and Omics Analysis, Guangming Advanced Research Institute and Department of Chemistry, School of Science, Southern University of Science and Technology, Shenzhen, 518055, China; Department of Chemical and Biological Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China.
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 signals using Zeno activation. To achieve cell type-resolved spatial proteomics, we developed a workflow integrating image-guided laser capture microdissection for the analysis of gastric cancer tissue. A 30-minute nanoLC gradient facilitated the quantification of 2,514 proteins from 0.2 mm2 regions representing cancer cells, cancer-associated fibroblasts (CAFs), and immune cells, as defined by multiplex immunohistochemistry (mIHC). This spatial proteomics approach revealed cellular heterogeneity within the tumor microenvironment of a clinical gastric cancer sample, and targeted validation identified the differential expression of 12 cancer-associated proteins comprising 103 peptides between cancer cell-enriched and CAF-enriched regions. This workflow offers a practical strategy for spatial proteomic analysis of clinical tissue samples.
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