Spatial proteome profiling by immunohistochemistry-based laser capture microdissection and data-independent acquisition proteomics

Peiwu Huang, Qian Kong, Weina Gao, Bizhu Chu, Hua Li, Yiheng Mao, Zongwei Cai, Ruilian Xu, Ruijun Tian*

*Corresponding author for this work

Research output: Contribution to journalJournal articlepeer-review

24 Citations (Scopus)


Understanding the tumor heterogeneity through spatially resolved proteome profiling is important for biomedical research and clinical application. Laser capture microdissection (LCM) is a powerful technology for exploring local cell populations without losing spatial information. Conventionally, tissue sections are stained with hematoxylin and eosin (H&E) for cell-type identification before LCM. However, it generally requires experienced pathologists to distinguish different cell types, which limits the application of LCM to broad cancer research field. Here, we designed an immunohistochemistry (IHC)-based workflow for cell type-resolved proteome analysis of tissue samples. Firstly, targeted cell type was marked by IHC using antibody targeting cell-type specific marker to improve accuracy and efficiency of LCM. Secondly, to increase protein recovery from chemically crosslinked IHC tissues, we optimized a decrosslinking procedure to seamlessly combine with the integrated spintip-based sample preparation technology SISPROT. This newly developed approach, termed IHC-SISPROT, has comparable performance as H&E staining-based proteomic analysis. High sensitivity and reproducibility of IHC-SISPROT were achieved by combining with data independent acquisition proteomics. More than 3500 proteins were identified from only 0.2 mm2 and 12 μm thickness of hepatocellular carcinoma (HCC) tissue section. Furthermore, using 5 mm2 and 12 μm thickness of HCC tissue section, 6660 and 6052 protein groups were quantified from cancer cells and cancer-associated fibroblasts (CAFs) by the IHC-SISPROT workflow. Bioinformatic analysis revealed the enrichment of cell type-specific ligands and receptors and potentially new communications between cancer cells and CAFs by these signaling proteins. Therefore, IHC-SISPROT is a sensitive and accurate proteomic approach for spatial profiling of cell type-specific proteome from tissues.

Original languageEnglish
Pages (from-to)140-148
Number of pages9
JournalAnalytica Chimica Acta
Publication statusPublished - 29 Aug 2020

Scopus Subject Areas

  • Analytical Chemistry
  • Biochemistry
  • Environmental Chemistry
  • Spectroscopy

User-Defined Keywords

  • Data-independent acquisition
  • Immunohistochemistry
  • Integrated sample preparation
  • Laser capture microdissection
  • Proteomics


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