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Enhanced Spatially Resolved Proteomics Using On-Tissue Hydrogel-Mediated Protein Digestion
David G Rizzo1, Boone M Prentice1, Jessica L Moore1
1Department of Chemistry, ‡Department of Biochemistry, §Mass Spectrometry Research Center, and ∥Departments of Pharmacology and Medicine, Vanderbilt University , Nashville, Tennessee 37232, United States.
Analytical Chemistry
|February 15, 2017
Summary
New hydrogel fabrication enables spatially resolved proteomic analysis of smaller tissue areas. This advancement allows for the identification of over 600 proteins from 260 μm hydrogels, revealing unique proteins in sub-regions.
Area of Science:
- Proteomics
- Biotechnology
- Materials Science
Background:
- Protein identification from tissue specimens often loses spatial information.
- Current spatially localized protein extraction using hydrogels is limited to 1-2 mm diameter areas.
- There is a need for smaller hydrogels to analyze smaller cell populations.
Purpose of the Study:
- To develop hydrogel fabrication processes for smaller diameter hydrogels (down to ~260 μm).
- To optimize hydrogel parameters (polyacrylamide concentration, trypsin loading) for enhanced protein identification.
- To demonstrate the utility of small hydrogels for spatially resolved proteomic analysis of tissue sub-regions.
Main Methods:
- Hydrogel fabrication processes were optimized for diameters as small as ~260 μm.
- Polyacrylamide concentrations (e.g., 18%) and trypsin loading were investigated.
- Liquid chromatography tandem mass spectrometry (LC-MS/MS) was used for protein identification.
Main Results:
- Hydrogel fabrication enabled applications down to ~260 μm diameter, a 15-fold reduction in area.
- Over 600 protein identifications were achieved from 260 μm hydrogels.
- Analysis of rat cerebellum sub-regions revealed ~20% unique proteins per region, demonstrating spatial resolution.
Conclusions:
- Improved hydrogel fabrication allows for robust, spatially localized proteomic analysis of smaller biological features.
- This technology facilitates targeted analysis of tissue subtypes for accurate characterization.
- The ability to analyze smaller regions enhances the understanding of tissue biology.
