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Ongoing advancements in instrumentation has established mass spectrometry (MS) as an essential tool in proteomics research and drug discovery. The newly released Asymmetric Track Lossless (Astral) analyzer represents a major step forward in MS instrumentation. Here, we evaluate the Orbitrap Astral mass spectrometer in the context of tandem mass tag (TMT)-based multiplexed proteomics and activity-based proteome profiling, highlighting its sensitivity boost relative to the Orbitrap Tribrid platform-50% at the peptide and 20% at the protein level. We compare TMT data-dependent acquisition and label-free data-independent acquisition on the same instrument, both of which quantify over 10,000 human proteins per sample within 1 h. TMT offers higher quantitative precision and data completeness, while data-independent acquisition is free of ratio compression and is thereby more accurate. Our results suggest that ratio compression is prevalent with the high-resolution MS2-based quantification on the Astral, while real-time search-based MS3 quantification on the Orbitrap Tribrid platform effectively restores accuracy. Additionally, we benchmark TMT-based activity-based proteome profiling by interrogating cysteine ligandability. The Astral measures over 30,000 cysteines in a single-shot experiment, a 54% increase relative to the Orbitrap Eclipse. We further leverage this remarkable sensitivity to profile the target engagement landscape of FDA-approved covalent drugs, including sotorasib and adagrasib. We herein provide a reference for the optimal use of the advanced MS platform.
He et al. (Wed,) studied this question.