Abstract
Spatial proteomics holds great potential to transform our understanding of the role of cell populations and their spatial organizations in human tissues related to diseases. However, in-depth proteome measurement with high spatial resolution has been challenged by the low sample input and inefficient proteomics workflow. In this protocol, we outlined the detailed procedure for high-resolution analysis of human pancreas tissue sections using a spatial proteomic platform by combining laser capture microdissection (LCM), nanoPOTS (nanodroplet processing in one pot for trace samples), and nanoflow liquid chromatography mass spectrometry (LC-MS). This protocol has been applied to human pancreas specimens for HubMAP Tissue Mapping Centers. Expected outcome: The unbiased spatial proteome mapping of >3000 proteins at 50-µm spatial resolution depending on sample types and LC-MS instrumentation. 1. Reagents 1.1. LCM collection • Dimethyl sulfoxide (DMSO) 1.2. Proteomic sample processing • Nanopure water • n-Dodecy-ß-D-maltoside (DDM) (e.g. Thermo Fisher Scientific Cat# 89902) • HEPES (pH 8.5) • TCEP (tris(2-carboxyethyl)phosphine)-HCL (Thermo Fisher scientific, Cat# A35349) • Chloroacetamide (CAA) (Thermo Fisher Scientific, Cat# A39270) • Lys-C, (Promega, cat. no. V1671) • Trypsin, (Promega, cat. no. V5280) • Formic acid
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