Spatial top-down proteomics for the functional characterization of human kidney

Area of Science:

• Proteomics
• Molecular Biology
• Biochemistry

Background:

• The Human Proteome Project has identified most predicted human proteins.
• The human proteome is complex, with millions of unique proteoforms due to various modifications.
• Mass spectrometry has begun mapping the proteoform landscape in bulk analyses.

Purpose of the Study:

• To develop an integrated workflow for characterizing proteoforms in human tissue with spatial resolution.
• To enable direct identification and spatial distribution mapping of proteoforms.

Main Methods:

• Coupling laser capture microdissection, nanoliter-scale sample preparation (microPOTS), and mass spectrometry imaging.
• Utilizing healthy human kidney sections for case study, focusing on glomeruli, tubules, and medullary rays.
• Performing sensitive top-down proteomics for proteoform measurement and library creation.

Main Results:

• Generated a quantitative database of 616 proteoforms from kidney tissue.
• Identified differential abundance of mitochondrial proteoforms between glomeruli and convoluted tubules.
• Mass spectrometry imaging confirmed microPOTS findings and revealed unique distributions, like truncated ubiquitin in cortical regions.

Conclusions:

• The developed workflow successfully identifies proteoforms and their spatial distributions.
• Distinct proteoform profiles were observed: mitochondrial proteoforms in tubules and hemoglobin subunits in glomeruli.
• This spatially resolved proteoform characterization can reshape discovery proteomics and aid in understanding disease etiology and pathophysiology.