Bait Correlation Improves Interactor Identification by Tandem Mass Tag-Affinity Purification-Mass Spectrometry

Area of Science:

• Proteomics
• Biochemistry
• Molecular Biology

Background:

• Quantitative multiplexing with isobaric tandem mass tags (TMT) enhances affinity purification mass spectrometry (AP-MS) throughput for protein interaction studies.
• Variable protein (bait) levels across experimental replicates complicate the accurate identification of interacting proteins.

Purpose of the Study:

• To compare Student's t-test and Pearson's R correlation for generating t-statistics in TMT-AP-MS.
• To assess the significance of protein interactors under varying experimental conditions.

Main Methods:

• Simulated protein recovery using a linear model to generate reporter ion ratio distributions.
• Experimental validation using two conditions: irreversible prey association (DNAJB8^H31Q) and reversible prey association (14-3-3ζ).
• Comparison of t-statistics derived from Student's t-test versus Pearson's R correlation.

Main Results:

• Correlation-derived t-statistics effectively mitigate the impact of bait variance and control false positives.
• Both methods performed comparably, but correlation-derived statistics showed significant improvement with high bait-level variance.
• Varying bait levels did not enhance selectivity but increased inter-run robustness.

Conclusions:

• Correlation-derived t-statistics offer a robust approach for interactor identification in TMT-AP-MS.
• This method enhances the reliability of protein interaction network characterization, especially in experiments with variable bait expression.
• The findings advocate for the adoption of correlation-based statistics to improve TMT-AP-MS data analysis.