Laser capture microdissected functional tissue units from kidney for top-down proteomic characterization using microPOTS

ABSTRACT: Human kidney tissue obtained from Vanderbilt University Biomolecular Multimodal Imaging Center (BIOMIC) was laser capture microdissected onto microPOTS chips. Six replicates of 100,000 um2 from three distinct functional tissue units (glomeruli, tubules, and medullary rays) were acquired from a 10 um thick kidney section. Each microPOTS well had a diameter of 2.2 mm and was preloaded with 2 uL of DMSO as the capture liquid. DMSO was evaporated by heating the chip at 70C and protein extraction was performed by adding 2 uL lysis buffer in each well, containing 2.5 units/uL benzonase nuclease, 2 mM MgCl2, 10 mM TCEP, 0.2% DDM and 4 M urea in 50 mM ABC. The mixture was incubated for 1 hr at 37C. Following incubation, the sample was acidified by adding 500 nL of 5% formic acid into each well and dried thoroughly in a vacuum chamber where the chips were stored at -20C until LC-MS/MS analysis. Samples were analyzed with an Orbitrap Fusion Lumos mass spectrometer in intact protein mode and data was searched using TopPIC (version 1.4) before downstream analysis in R with the TopPICR R package

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Ljiljana Pasa-Tolic  

PROVIDER: MSV000093722 | MassIVE | Fri Dec 22 13:24:00 GMT 2023

REPOSITORIES: MassIVE