Towards high spatially resolved microproteomics using expansion microscopy

Abstract

Expansion microscopy 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 folds 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 to carry out MS-based proteomic analysis. A major issue in the Expansion microscopy is the loss of proteins information during the mechanical homogenization phase due to the use of Proteinase K. For isotropic expansion, different homogenization agents are investigated, both to maximize protein identification and to minimize protein diffusion. Better results are obtained with SDS. From a tissue section enlarge more than 3-fold, we have been able to manually cut out regions of 1mm in size, equivalent to 300{micro}m in their real size. We identified up to 655 proteins from a region corresponding to an average of 940 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. Abstract graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/433765v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@14cc519org.highwire.dtl.DTLVardef@1201e36org.highwire.dtl.DTLVardef@19fa853org.highwire.dtl.DTLVardef@e4112a_HPS_FORMAT_FIGEXP M_FIG C_FIG