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

Area of Science:

• Proteomics
• Biochemistry
• Molecular Biology

Background:

• The human proteome comprises millions of unique proteoforms due to various biological processes.
• Current bulk analysis methods using mass spectrometry have limitations in spatial resolution.
• A need exists for spatially resolved proteoform characterization in human tissues.

Purpose of the Study:

• To develop and validate an integrated workflow for characterizing proteoforms in human tissue with spatial resolution.
• To map the spatial distribution of proteoforms within functional units of the human kidney.
• To identify differentially abundant proteoforms between kidney glomeruli and tubules.

Main Methods:

• Coupling laser capture microdissection with nanoliter-scale sample preparation (microPOTS) for top-down proteomics.
• Utilizing mass spectrometry imaging for near-cellular spatial resolution.
• Developing a quantitative proteoform database for library-based imaging.

Main Results:

• Successfully mapped 616 proteoforms in human kidney tissue with near-cellular resolution.
• Identified distinct proteoform profiles in glomeruli (hemoglobin subunits) and tubules (mitochondrial proteoforms).
• Observed differential abundance of specific proteoforms, including a truncated ubiquitin form in cortical regions.

Conclusions:

• The developed workflow enables direct identification and spatial mapping of proteoforms.
• Spatially resolved proteoform data can reshape discovery-based proteomics by linking proteoforms to cellular functions.
• This technology holds potential for unraveling disease etiology and pathophysiology.