Protein-Protein Interactions with Yeast Two-Hybrid and Mass Spectrometry

Introduction to Protein-Protein Interactions

Protein-protein interactions (PPIs) are fundamental to almost all biological processes and govern the majority of cellular pathways. Understanding these interactions is crucial for unraveling the complex network of biochemical pathways in organisms. Various technologies have been developed to systematically map these interactions, each with their own strengths and challenges.

Yeast Two-Hybrid System

The yeast two-hybrid (Y2H) system is a widely used method for studying PPIs. This technique, developed over 25 years ago, remains one of the most cost-effective, time-saving, and straightforward methods for identifying and analyzing PPIs. The Y2H assay is versatile, enabling the identification of interacting partners for a given protein, verifying interactions between known proteins, and mapping interacting domains. The majority of interactions identified through this method are binary direct interactions.

Mass Spectrometry in PPI Analysis

Mass spectrometry has become an essential tool in the study of PPIs, particularly in understanding the architecture of multi-protein complexes. Crosslinking mass spectrometry (Crosslinking MS) provides distance restraints between protein residues, offering insights into the topology of protein interactions. This method, traditionally used on purified protein complexes, is now being applied to more complex systems, such as cell lysates, organelles, and whole cells.

Error Estimation in Mass Spectrometry

A significant challenge in using Crosslinking MS for mapping PPIs is accurately estimating the false discovery rate (FDR). To ensure the reliability of reported interactions, it's crucial to correctly estimate FDRs and trim the list of reported PPIs to the desired error rate. The target-decoy approach is a standard method for error estimation in Crosslinking MS, but it requires careful handling, especially when considering different types of crosslinks (heteromeric and self-links) and the propagation of error through different levels of data analysis.

Improving PPI Reliability in Crosslinking Mass Spectrometry

Recent research has focused on refining error estimation methods in Crosslinking MS to improve the reliability of PPI data. Experimental controls and innovative approaches in data analysis have been employed to address the inflated error of naive decoy-based FDRs. By considering heteromeric matches separately from self-matches and focusing on error propagation between information levels, researchers have achieved more accurate PPI error estimations, thus enhancing the reliability of Crosslinking MS data.

Advances and Future Directions

There have been significant advancements in the enrichment and detection of crosslinked peptides, suggesting that Crosslinking MS will soon be capable of mapping large portions of the cellular interactome in a single experiment. These developments will enable researchers to detect changes in interactomes across different cellular states, a crucial step forward in understanding the dynamic nature of PPIs.

Conclusion

In summary, the study of protein-protein interactions using techniques like the yeast two-hybrid system and crosslinking mass spectrometry has greatly advanced our understanding of cellular processes. Continuous improvements in these methods, especially in data accuracy and error estimation, are crucial for providing a more comprehensive and reliable picture of the protein interaction networks that drive life at the molecular level.