TMT-Based Multiplexed (Chemo)Proteomics on the Orbitrap Astral Mass Spectrometer

Area of Science:

• Proteomics
• Mass Spectrometry
• Drug Discovery

Background:

• Mass spectrometry (MS) is crucial for proteomics and drug discovery.
• Advancements in MS instrumentation are continuously improving research capabilities.
• The Orbitrap Astral analyzer is a new, powerful MS instrument.

Purpose of the Study:

• Evaluate the Orbitrap Astral mass spectrometer for TMT-based multiplexed proteomics and activity-based proteome profiling.
• Compare Astral's sensitivity and performance against the Orbitrap Tribrid platform.
• Benchmark TMT-based activity-based proteome profiling for cysteine ligandability and drug target engagement.

Main Methods:

• Tandem Mass Tag (TMT)-based multiplexed proteomics.
• Activity-based proteome profiling (ABPP).
• Data-dependent acquisition (DDA) and label-free data-independent acquisition (DIA) on the Astral.
• Comparison with Orbitrap Tribrid and Orbitrap Eclipse platforms.

Main Results:

• The Astral shows a 50% peptide and 20% protein sensitivity boost over the Tribrid platform.
• Both TMT-DDA and LF-DIA quantify over 10,000 human proteins per sample within 1 hour.
• Astral measures over 30,000 cysteines in a single-shot, a 54% increase over the Eclipse.
• Ratio compression observed in Astral's MS2 quantification; MS3 on Tribrid restores accuracy.

Conclusions:

• The Orbitrap Astral offers enhanced sensitivity for proteomics and proteome profiling.
• TMT-based DIA and LF-DIA provide complementary quantitative insights.
• Astral's sensitivity is valuable for interrogating cysteine ligandability and drug target engagement.