Full-Featured, Real-Time Database Searching Platform Enables Fast and Accurate Multiplexed Quantitative Proteomics

Area of Science:

• Proteomics
• Mass Spectrometry
• Computational Biology

Background:

• Multiplexed quantitative proteomic analyses are crucial for biological insights.
• The gold standard, SPS-MS3, has long acquisition cycles hindering efficiency.
• Need for faster, accurate methods in complex proteome analysis.

Purpose of the Study:

• To develop a real-time database search (RTS) platform, Orbiter, to overcome SPS-MS3's limitations.
• To improve the efficiency and accuracy of multiplexed quantitative proteomic experiments.
• To enable faster and more comprehensive protein quantification.

Main Methods:

• Developed Orbiter, a real-time database search (RTS) platform.
• Integrated RTS with Comet for peptide spectral matching and false discovery rate analysis.
• Implemented adaptive data acquisition triggered by spectral match results.

Main Results:

• Orbiter processed single spectrum database searches in under 10 ms.
• Achieved a 2-fold increase in mass spectrometric data acquisition efficiency.
• Quantified over 8000 proteins across 10 proteomes in 18 hours, half the time of SPS-MS3.

Conclusions:

• Orbiter's RTS platform significantly enhances proteomic analysis speed and efficiency.
• The method allows for accurate quantification and identification of post-translational modifications.
• This adaptive data acquisition strategy improves throughput for complex proteome studies.