# Electroblotting through enzymatic membranes to enhance MALDI tissue imaging of proteins

> **NIH NIH R21** · OHIO STATE UNIVERSITY · 2020 · $224,405

## Abstract

Project Summary
This project aims to develop a powerful new approach to improve the spatially resolved
extraction and identification of proteins from tissue. Detection of proteins for imaging mass
spectrometry analysis is challenging because of limited ionization. To address this issue,
electrophoresis will be employed to extract proteins from tissues and into adjacent
enzymatic membranes for proteolytic digestion. The resulting peptides will be collected on
a capture membrane for Matrix Assisted Laser/Desorption Ionization Imaging Mass
Spectrometry (MALDI-IMS) analysis. This approach both increases the number of proteins
that can be identified from tissues and preserves their spatial distribution in the original
tissue.
The project is constructed around three sets of activities. First, the spatial diffusion of the
resulting peptides will be measured after they are digested in the enzymatic membrane
under a range of experimental conditions. A standard mixture of proteins will be separated
by gel electrophoresis and then the proteins will be electrophoresed in an orthogonal
direction, through an enzymatic membrane. The digested peptides will be collected on a
capture membrane for mass spectrometry analysis and compared against optical images
of the protein gels prior to electrodigestion to determine the lateral diffusion. A range of
experimental conditions will be evaluated, including voltages, membrane pore sizes and
denaturants, to determine the optimal conditions. These optimized conditions will be
applied to the study of proteins in mouse brains in the second set of experiments. In the
third and final set of experiments, pepsin membranes will be added to the workflow to
increase proteomic coverage.
The public health benefits of the project lie in the promise of a powerful new tool to analyze
protein expression in tissues for both diagnostic purposes and mechanistic studies. This
approach will make it possible for researchers to build a coherent picture of the protein
expression levels that underlie the development of diseases, thus improving patient
outcomes.

## Key facts

- **NIH application ID:** 9861203
- **Project number:** 5R21AG062144-02
- **Recipient organization:** OHIO STATE UNIVERSITY
- **Principal Investigator:** Amanda B. Hummon
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $224,405
- **Award type:** 5
- **Project period:** 2019-02-15 → 2022-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9861203

## Citation

> US National Institutes of Health, RePORTER application 9861203, Electroblotting through enzymatic membranes to enhance MALDI tissue imaging of proteins (5R21AG062144-02). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/9861203. Licensed CC0.

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