Sensitive and Accurate Proteome Profiling of Embryogenesis Using Real-Time Search and TMTproC Quantification

Area of Science:

• Proteomics
• Systems Biology
• Developmental Biology

Background:

• Multiplexed proteomics is vital for biological system investigation.
• TMTproC complementary ions address ratio distortion but require long acquisition times.
• Real-time search (RTS) algorithms improve speed and sensitivity in proteomics.

Purpose of the Study:

• To combine TMTproC complementary ion quantification with RTS (TMTproC-RTS).
• To enhance sensitivity, accuracy, and precision in MS2-level quantitative proteomics.
• To analyze protein dynamics during early embryogenesis in diverse model organisms.

Main Methods:

• Integration of TMTproC complementary ion quantification with real-time search (RTS).
• Application of the TMTproC-RTS method to quantify protein dynamics in embryonic development.
• Utilizing Orbitrap mass spectrometry for high-resolution quantitative analysis.

Main Results:

• Demonstrated enhanced sensitivity and accuracy in MS2-level quantitative proteomics.
• Quantified 7.8k (fly), 8.6k (sea squirt), and 12.7k (frog) proteins, a 12-14% improvement over naive TMTproC.
• Generated deep and accurate protein dynamics datasets for three model organisms.

Conclusions:

• TMTproC-RTS significantly improves protein quantification in multiplexed proteomics.
• The method provides valuable insights into evolutionary comparisons of early embryogenesis.
• This work establishes a robust dataset for studying developmental protein dynamics.