Benchmarking the Orbitrap Tribrid Eclipse for Next Generation Multiplexed Proteomics

Area of Science:

• Proteomics
• Mass Spectrometry
• Quantitative Biology

Background:

• Quantitative proteomics is crucial for dissecting complex biological phenotypes.
• Advancements in instrumentation and software are key to improving proteomic analysis.
• Sample multiplexing strategies are increasingly used in comparative studies.

Purpose of the Study:

• To evaluate the utility of the Orbitrap Eclipse Tribrid mass spectrometer and its new software features for quantitative proteomics.
• To assess the impact of specific features like Precursor Fit filtering, TurboTMT, and Real-time Peptide Search on data acquisition and analysis.
• To determine if enhanced sensitivity and identification rates affect quantitative accuracy.

Main Methods:

• Utilized the Orbitrap Eclipse Tribrid mass spectrometer with advanced quadrupole filter and optimized FTMS scan overhead.
• Implemented new instrument control software features: Precursor Fit filtering, TurboTMT, and Real-time Peptide Search filtering.
• Performed multidimensional comparisons of these novel features using SPS-MS3 and HRMS2 methods.

Main Results:

• Achieved a 20% increase in total peptide identifications for SPS-MS3 methods.
• Observed a 14% increase in total peptide identifications for HRMS2 methods.
• Real-time Peptide Search filtering resulted in approximately a 2x throughput improvement for quantification without sacrificing accuracy.

Conclusions:

• The evaluated hardware and software features enhance the efficiency and depth of quantitative proteomics.
• These advancements facilitate more comprehensive comparative proteome-wide insights.
• The new capabilities support more robust dissection of complex phenotypic differences.