Toward High Spatially Resolved Proteomics Using Expansion Microscopy

Expansion
microscopy (EM) is an emerging approach for morphological
examination of biological specimens at nanoscale resolution using
conventional optical microscopy. To achieve physical separation of
cell structures, tissues are embedded in a swellable polymer and expanded
several fold in an isotropic manner. This work shows the development
and optimization of physical tissue expansion as a new method for
spatially resolved large-scale proteomics. Herein we established a
novel method to enlarge the tissue section to be compatible with manual
microdissection on regions of interest and MS-based proteomic analysis.
A major issue in expansion microscopy is the loss of protein information
during the mechanical homogenization phase due to the use of proteinase
K. For isotropic expansion, different homogenization agents were investigated,
both to maximize protein identification and to minimize protein diffusion.
Best results were obtained with SDS for homogenization. Using our
modified protocol, we were able to enlarge a tissue section more than
3-fold and identified up to 655 proteins from 1 mm in size after expansion,
equivalent to 330 μm in their real size corresponding thus to
an average of 260 cells. This approach can be performed easily without
any expensive sampling instrument. We demonstrated the compatibility
of sample preparation for expansion microscopy and proteomic study
in a spatial context.