Zhen Dong (Hangzhou / CN), Wenhao Jiang (Hangzhou / CN), Jiayi Chen (Hangzhou / CN), Chunlong Wu (Hangzhou / CN), Pingping Hu (Hangzhou / CN), Ting Chen (Hangzhou / CN), Xuan Ding (Hangzhou / CN), Shu Zheng (Hangzhou / CN), Kiryl Piatkevich (Hangzhou / CN), Yi Zhu (Hangzhou / CN), Tiannan Guo (Hangzhou / CN)
Introduction
Understanding tissue heterogeneity is crucial for studying disease evolution and drug targets. High-throughput spatial proteomics with subcellular resolution can significantly advance this field. Current antibody-based techniques like CODEX offer subcellular resolution but are limited in throughput and antibody availability, hindering new protein biomarker discovery. Mass spectrometry (MS) methods like MALDI-MS do not require antibodies but can only characterize a limited number of peptides and proteins. Improvements in MS instruments have made it feasible to analyze minute tissue samples via laser microdissection (LCM), as demonstrated by methods such as LCM-nanoPOTs and LCM-DVP. However, LCM-based methods may compromise sample quality, rely heavily on expertise, and face technical bottlenecks for samples with resolutions below 10-20 µm.
Methods
Here we present a spatial proteomics technology, termed filter-aided expansion proteomics (FAXP), using hydrogel-based tissue expansion and data-independent acquisition (DIA) MS for proteomics of formalin-fixed paraffin-embedded (FFPE) specimens. The FFPE tissue sections are embedded in customized hydrogel and can be expanded by a linear scale of 4-5 folds. After expansion, a minute tissue area, down to near single cell level, could be manually dissected and subject to DIA-based MS analysis.
To achieve subcellular resolution, we further enhance FAXP by combining iterative tissue expansion with super-resolution imaging, LCM, robotics, and advanced MS techniques. This combination overcomes the resolution limits of LCM, enabling spatial subcellular proteomics analysis.
Results
Compared to our original ProteomEx method1, FAXP, now partially automated via robotics, achieves a 14.5-fold increase in volumetric resolution of sample preparation. It generates a higher peptide yield, resulting in a 250% rise in protein identifications, while reducing sample preparation time by 50%, leading to identification of over 6700 proteins from FFPE specimens containing different stages of malignant changes of colorectal adenomas at specific positions.
Building on FAXP, we iteratively expand cells and tissue samples from 512-fold to over 8000-fold in volume. This new strategy enables sub-100 nm super-resolution imaging using conventional confocal microscopes (e.g., Zeiss 980). Assisted by super-resolution imaging, we have delineated intricate subcellular organelle structures and identified nearly 2000 proteins from a single bona fide nucleus. Next, we plan to map smaller organelles like mitochondria and analyze them in AD mouse models for key factors.
Conclusions
A novel spatial proteomics technology, FAXP, has been developed and implemented on clinical FFPE samples, enabling subcellular analysis guided by imaging. In this presentation, we will discuss its technical details, utility, pros, and cons.
1. Li, L. et al. Spatially resolved proteomics via tissue expansion. Nat. Commun. 13, 7242 (2022).
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