Spatially Resolved Top-Down Proteomics of Tissue Sections Based on a Microfluidic Nanodroplet Sample Preparation Platform

Conventional proteomic approaches measure the aver-aged signal from mixed cell populations or bulk tissues, leading to the dilution of signals arising from sub -populations of cells that might serve as important bio-markers. Recent developments in bottom-up proteomics have enabled spatial mapping of cellular heterogeneity in tissue microenvironments. However, bottom-up prote-omics cannot unambiguously define and quantify proteo-forms, which are intact (i.e., functional) forms of proteins capturing genetic variations, alternatively spliced tran-scripts and posttranslational modifications. Herein, we described a spatially resolved top-down proteomics (TDP) platform for proteoform identification and quantitation directly from tissue sections. The spatial TDP platform consisted of a nanodroplet processing in one pot for trace samples-based sample preparation system and an laser capture microdissection-based cell isolation system. We improved the nanodroplet processing in one pot for trace samples sample preparation by adding benzonase in the extraction buffer to enhance the coverage of nucleus proteins. Using similar to 200 cultured cells as test samples, this approach increased total proteoform identifications from 493 to 700; with newly identified proteoforms primarily corresponding to nuclear proteins. To demonstrate the spatial TDP platform in tissue samples, we analyzed laser capture microdissection-isolated tissue voxels from rat brain cortex and hypothalamus regions. We quantified 509 proteoforms within the union of top-down mass spectrometry-based proteoform identification and char-acterization and TDPortal identifications to match with features from protein mass extractor. Several proteoforms corresponding to the same gene exhibited mixed abun-dance profiles between two tissue regions, suggesting potential posttranslational modification-specific spatial distributions. The spatial TDP workflow has prospects for biomarker discovery at proteoform level from small tissue sections.

Automated summary

Conventional proteomic approaches cannot accurately measure signals from sub-populations of cells, while recent developments in bottom-up proteomics enable spatial mapping of cellular heterogeneity. However, bottom-up proteomics cannot define and quantify intact forms of proteins capturing genetic variations and posttranslational modifications. A spatially resolved top-down proteomics (TDP) platform is described, which allows direct identification and quantitation of proteoforms from tissue sections. This platform consists of a nanodroplet processing system for sample preparation and a laser capture microdissection-based cell isolation system. The improved sample preparation method enhances the coverage of nuclear proteins. The TDP platform has potential for biomarker discovery at the proteoform level from small tissue sections.