With each passing year, advances in cellular resolution continue to accelerate, enabling discoveries at increasingly smaller scales. Now, the Filter-Aided eXpansion Proteomics (FAXP) workflow emerges as a novel and innovative method for resolving the molecular complexity of cellular structures.

Developed by Dong et al. at Westlake University (China) and recently published in Nature Communications, FAXP integrates hydrogel-based tissue expansion techniques with mass spectrometry (MS) to advance the field of spatial proteomics research, with broad potential applications, particularly in research and personalised medicine.

Building upon their earlier proteomics method, ProteomX, Dong et al. demonstrate how FAXP enables high-throughput processing without the traditional limitations of sample size, significantly improving spatial resolution and efficiency. A key highlight of FAXP is its simplicity, differing subtly from ProteomX while maintaining the same number of steps. However, these refinements result in major efficiency gains: sample anchoring time is reduced by more than 90%, contributing to a 50% reduction in overall sample preparation time while also increasing reproducibility.

The new workflow introduces key enhancements to the preparation protocol following tissue sample collection, ensuring effective sample anchoring and isotropic expansion before staining and laser capture microdissection (LCM). Additionally, the integration of filter-aided in-gel digestion minimises the risk of sample loss during pipetting.

Validation of the workflow was conducted across digested sections of nuclei, cells, and blank controls, analysed using a Thermo Orbitrap Astral mass spectrometer interfaced with a Vanquish™ Neo UHPLC system, utilising an IonOpticks Aurora Elite XT 15 × 75 C18 UHPLC column for peptide separation. The results demonstrated a 14.5-fold increase in volumetric resolution, an 8-fold rise in peptide yield, and over 255% more protein identifications compared to previous methods. Additionally, FAXP successfully enabled the analysis of proteins in FFPE-archived colorectal tumor samples, overcoming key limitations of traditional proteomics approaches in this challenging matrix.              

Furthermore, the optimised hydrogel-based tissue expansion method integrated with LC-MS enables the isolation and study of proteins at subcellular resolution. By leveraging AI-driven image analysis, laser capture microdissection, and automated tissue handling, FAXP allows researchers to pinpoint regions of interest with precision while cutting sample preparation times in half. Together, these innovations make high-resolution proteomics more accessible and pave the way for applications in cancer research, biomarker discovery, and precision medicine.

Publication
Nature Communications

Authors

et, al.

Title

Spatial proteomics of single cells and organelles on tissue slides using filter-aided expansion proteomics