High-Throughput Large-Scale Targeted Proteomics Assays for Quantifying Pathway Proteins in Pseudomonas putida KT2440

Area of Science:

• Proteomics
• Systems Biology
• Metabolic Engineering

Background:

• Targeted proteomics using liquid chromatography-selected reaction monitoring (LC-SRM) is vital for systems biology and metabolic engineering.
• High-throughput analysis is crucial for large sample sets in systems biology.
• Nanoflow LC-SRM offers sensitivity, but microflow LC-SRM provides higher speed.

Purpose of the Study:

• To evaluate the performance of microflow LC-SRM compared to nanoflow LC-SRM for high-throughput targeted proteomics.
• To assess if microflow LC-SRM can maintain sensitivity and multiplexing power with recent mass spectrometry advancements.
• To determine the suitability of microflow LC-SRM for analyzing large sample sets in systems biology.

Main Methods:

• Comparative analysis of microflow and nanoflow LC-SRM platforms.
• Monitoring of 339 peptides from 132 enzymes in *Pseudomonas putida* KT2440.
• Response curve studies on 248 peptides to assess sensitivity and stability.

Main Results:

• Highly correlated results between microflow and nanoflow LC-SRM platforms.
• Microflow LC-SRM demonstrated comparable sensitivity to nanoflow LC-SRM for most peptides.
• Microflow LC-SRM exhibited superior mass spectrometry signal and chromatography stability.

Conclusions:

• Microflow LC-SRM is a viable high-throughput alternative to nanoflow LC-SRM for targeted proteomics.
• The speed of microflow LC-SRM can be leveraged without significant loss of sensitivity or multiplexing capability.
• Microflow LC-SRM enhances data quality through improved signal and chromatography stability, benefiting systems biology research.