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Comprehensive Workflow of Mass Spectrometry-based Shotgun Proteomics of Tissue Samples
Published on: November 13, 2021
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Tissue proteomics using chemical immobilization and mass spectrometry
Punit Shah1, Bai Zhang1, Caitlin Choi1
1Department of Pathology, Johns Hopkins University, Baltimore, MD 21231, USA.
Analytical Biochemistry
|October 7, 2014
Summary
A new chemical immobilization method (CIPPE) effectively removes interference from optimal cutting temperature (OCT) medium during tissue proteomic analysis. This enables high-throughput mass spectrometry (MS) of frozen tissues, improving disease biomarker discovery.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Molecular Biology
Background:
- Proteomics analysis of tissues provides critical biological and pathological insights for disease diagnostics and therapy.
- Optimal cutting temperature (OCT) embedding, common for tissue preservation, and other sample components interfere with mass spectrometry (MS) based proteomic analysis.
- Existing methods struggle with high-throughput proteomic analysis of OCT-embedded tissues due to OCT interference.
Purpose of the Study:
- To develop and validate a novel method for overcoming interference in proteomic analysis of OCT-embedded tissues.
- To enable high-throughput, reproducible proteomic profiling of frozen tissue samples for disease research.
Main Methods:
- Chemical immobilization of proteins for peptide extraction (CIPPE) was developed, immobilizing proteins onto a solid support.
- Extensive washing removed tissue and OCT interferences from immobilized proteins before proteolysis and peptide release.
- The method was validated using human serum albumin to eliminate OCT contamination and applied to frozen OCT-embedded tissues with iTRAQ labeling and 2D LC-MS/MS.
Main Results:
- CIPPE successfully eradicated OCT-specific interference in standard protein analysis.
- Proteomic analysis of frozen OCT-embedded tissues yielded reproducible extraction and quantitation of 10,284 proteins (3996 protein groups).
- The method demonstrated minimal impact of OCT embedding on global proteome analysis of stored tissue samples.
Conclusions:
- CIPPE is an effective technique for high-throughput proteomic analysis of OCT-embedded tissues.
- This method facilitates the study of global tissue proteomes from preserved samples, aiding in disease biomarker identification.
- CIPPE overcomes a significant technical hurdle in tissue proteomics, enhancing its utility in biomedical research.
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