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Related Experiment Video

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Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation

Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation

Published on: August 21, 2019

Multiplexed Cross-Linking with Isobaric Quantitative Protein Interaction Reporter Technology.

Juan D Chavez¹, Andrew Keller¹, Helisa H Wippel¹

¹Department of Genome Sciences, University of Washington, Seattle, Washington 98195, United States.

Analytical Chemistry

|December 9, 2021

Summary

New multiplexed isobaric quantitative protein interaction reporter (iqPIR) cross-linkers enable simultaneous quantification of up to six samples in chemical cross-linking with mass spectrometry (XL-MS) studies.

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Area of Science:

Biochemistry and Molecular Biology
Analytical Chemistry
Proteomics

Background:

Chemical cross-linking with mass spectrometry (XL-MS) is vital for studying protein structures and interactions.
Quantitative XL-MS, using stable isotopes, reveals protein conformational and interaction dynamics.
Existing methods often rely on deuterium labeling and MS1-based quantitation, limiting multiplexing.

Purpose of the Study:

To develop and evaluate multiplexed isobaric quantitative protein interaction reporter (iqPIR) cross-linkers.
To enable simultaneous quantification of up to six cross-linked samples.
To adapt iqPIR quantitative analysis for multi-channel data with improved accuracy and reproducibility.

Main Methods:

Synthesis and evaluation of multiplexed iqPIR molecules utilizing ¹³C and ¹⁵N isotope labels.
Adaptation of two-channel iqPIR analysis to accommodate multiple channels with defined ion isotope peak mass offsets.
Development of a data analysis method to apportion summed ion peak intensities in overlapping isotope envelopes.

Main Results:

Successful synthesis and initial evaluation of multiplexed iqPIR reagents.
Accurate and reproducible relative quantitation of up to six differentially labeled cross-linked samples.
Demonstration of a robust data analysis pipeline for multi-channel iqPIR data.

Conclusions:

The developed multiplexed iqPIR strategy significantly enhances the scalability of quantitative XL-MS.
This approach allows for direct comparison across multiple samples, facilitating large-scale interactome studies.
The methodology is extensible for future iqPIR reagent designs and in vivo applications.