Related Experiment Video
Updated: Apr 15, 2026
07:44
TMT Sample Preparation for Proteomics Facility Submission and Subsequent Data Analysis
Published on: June 8, 2020
13.6K
Selective Proteomic Proximity Labeling Assay Using Tyramide (SPPLAT): A Quantitative Method for the Proteomic
Johanna Susan Rees1,2, Xue-Wen Li3, Sarah Perrett3
1Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom.
Current Protocols in Protein Science
|April 2, 2015
Summary
This study introduces a novel method to pinpoint proteins in specific cell membrane regions. It uses horseradish peroxidase (HRP) and a biotinylated reagent to label nearby proteins for identification via mass spectrometry.
Area of Science:
- Cell biology
- Proteomics
- Biochemistry
Background:
- Understanding protein localization is crucial for cell function.
- Existing methods may lack spatial resolution.
- Identifying proteins in membrane microenvironments is challenging.
Purpose of the Study:
- To develop a general method for identifying proteins in spatially restricted membrane microenvironments.
- To enable precise mapping of protein neighborhoods at the nanoscale.
Main Methods:
- Utilizes horseradish peroxidase (HRP) conjugated to a target-binding molecule.
- Employs a biotinylated tyramide reagent that is activated by HRP and hydrogen peroxide.
- The activated reagent generates a short-range free radical for proximity-dependent protein labeling.
- Labeled proteins are isolated using affinity chromatography and identified by liquid chromatography-mass spectrometry (LC-MS).
Main Results:
- Successfully labels proteins within tens to hundreds of nanometers of the target.
- Enables identification of proteins in specific membrane microenvironments.
- The method is adaptable for quantitative analysis using SILAC or isobaric tagging.
Conclusions:
- This technique provides a powerful tool for mapping protein interactions and localizations at the cell membrane.
- It offers high spatial resolution for studying cellular organization.
- The method is versatile and can be applied to various biological questions.
