Routine Workflow of Spatial Proteomics on Micro-formalin-Fixed Paraffin-Embedded Tissues

Anal Chem. 2023 Nov 14;95(45):16733-16743. doi: 10.1021/acs.analchem.3c03848. Epub 2023 Nov 3.

Abstract

In the era of single-cell biology, spatial proteomics has emerged as an important frontier. However, it still faces several challenges in technology. Formalin-fixed paraffin-embedded (FFPE) tissues are an important material in spatial proteomics, in which fixed tissues are excised using laser capture microdissection (LCM), followed by protein identification with mass spectrometry. For a satisfied spatial proteomics upon FFPE tissues, the excision area is expected to be as small as possible, and the identified proteins are countered upon as much as possible. For a general laboratory for spatial proteomics, a routine workflow is required, not relying on any special device, and is easily operating. In view of these challenges in technology, we initiated a technology evaluation throughout the entire procedure of proteomic analysis with micro-FFPE tissues. In contrast to the protocols reported previously, several innovations in technology were proposed and conducted, such as removal of destaining, decross-linking with "hang-down", solution simplification for peptide generation and balancing to excision area, and capture rate of micro-FFPE tissues. After optimization of all the necessary steps, a routine workflow was established, in which the minimized area for protein identification was 0.002 mm2, while the excision area for a consistent proteomic analysis was 0.05 mm2. Using the developed workflow and collecting the micro-FFPE tissues continuously, for the first time, a spatial proteomic atlas of mouse brain was preliminarily constructed, which exhibited the typical characteristics of spatial-dependent protein abundance and functional enrichment.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Formaldehyde* / chemistry
  • Mice
  • Paraffin Embedding / methods
  • Proteins / analysis
  • Proteomics* / methods
  • Tissue Fixation / methods
  • Workflow

Substances

  • Formaldehyde
  • Proteins