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Analyzing Large Protein Complexes by Structural Mass Spectrometry
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Published on: June 19, 2010

Analyzing protein-protein interactions by quantitative mass spectrometry.

Florian E Paul1, Fabian Hosp, Matthias Selbach

  • 1Cell Signaling and Mass Spectrometry Group, Max Delbrück Center for Molecular Medicine, Berlin, Germany.

Methods (San Diego, Calif.)
|March 9, 2011
PubMed
Summary

Quantitative affinity purification followed by mass spectrometry (q-AP-MS) is a powerful method for studying protein-protein interactions (PPIs). This study details two q-AP-MS protocols to investigate PPIs and protein function.

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A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation
14:44

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Published on: September 24, 2012

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Proteomics

Background:

  • Cellular processes rely on protein-protein interactions (PPIs), making their study crucial for understanding protein function.
  • Quantitative affinity purification followed by mass spectrometry (q-AP-MS) is an unbiased method for PPI investigation.
  • q-AP-MS enriches target proteins with their interactors and uses control experiments to identify non-specific binding.

Purpose of the Study:

  • To provide detailed protocols for quantitative affinity purification followed by mass spectrometry (q-AP-MS).
  • To enable researchers to adapt q-AP-MS for diverse biological questions concerning protein-protein interactions.
  • To discuss the benefits, limitations, and critical parameters of q-AP-MS.

Main Methods:

  • Presents two distinct q-AP-MS protocols: one using immobilized bait proteins and another employing transient transfection of tagged expression constructs.
  • Describes the quantitative mass spectrometry approach used to compare protein levels between pull-down and control samples.
  • Emphasizes the exclusion of non-specific contaminants through comparative analysis.

Main Results:

  • Detailed q-AP-MS protocols are provided for two common experimental setups.
  • Discussion highlights key parameters influencing the success of q-AP-MS experiments.
  • The presented information facilitates the application of q-AP-MS for various research needs.

Conclusions:

  • The provided protocols and background information empower researchers to implement q-AP-MS effectively.
  • This work supports the investigation of protein function through the study of protein-protein interactions.
  • Adaptable q-AP-MS strategies are essential for advancing biological research.