PNNL

Abstract

Multiplexed bimolecular profiling of tissue microenvironment, or spatial omics, can provide deep insight into cellular compositions and interactions in both normal and diseased tissues. Proteome-scale tissue mapping, which aims to unbiasedly visualize all the proteins in whole tissue section or region of interest, has attracted significant interest because it holds great potential to directly reveal diagnostic biomarkers and therapeutic targets. While many approaches available, however, proteome mapping still exhibits significant technical challenges in both protein coverages and analysis throughput. We reason most of these existing challenges are associated with mass spectrometry-based protein identification and quantification. Thus, we performed a detailed benchmarking study of three protein quantification methods for spatial proteome mapping, including label-free, TMT-MS2, and TMT-MS3. Our study indicates label-free method provided the deepest coverages of ~3500 proteins at a spatial resolution of 50 µm, while TMT-MS2 method holds great benefit in mapping throughput at >125 pixels per day. The evaluation also indicates both label-free and TMT-MS2 provide robust protein quantifications in term of identifying differentially abundant proteins and spatially co-variable clusters. In the study of pancreatic islet microenvironment, we demonstrated deep proteome mapping not only enables to identify protein markers specific to different cell types, more importantly, it also reveal unknown or hidden protein patterns by spatial co-expression analysis.