Novel data-independent acquisition and targeted quantitation on Orbitrap Tribrid instruments, unleashing the power of Hybrid-DIA technology

For any proteomic analysis using mass spectrometry, the desired outcome defines the methodology of choice. However, each methodology has tradeoffs, and relying on a single approach may be insufficient to the task. For example, rapid chromatography increases sample throughput, but limits the sensitivity and quantitative capabilities of the sample analysis. Tandem mass tagging of peptides multiplexes sample analysis, but requires high resolution and data-dependent methods. Targeted methods focus on a small set of peptides, leaving much of the sample unexplored. To overcome this challenge, sample analysis requires serially applied methodologies, a costly redundancy if sample or instrument availability is limited.

Hybrid-DIA MS technology offers a novel solution by intelligently integrating targeted peptide analysis and discovery-based data independent acquisition (DIA), simultaneously maximizing instrument utilization and increasing sample throughput. Hybrid-DIA MS operates through the Thermo Fisher Scientific Moonshot application, so far only available to the Exploris 480 Orbitrap mass spectrometer via the Instrument Application Programming Interface (IAPI).

We present Moonshot Pro, a redevelopment of the Moonshot application for the Tribrid series of Orbitrap mass spectrometers, with greatly improved functionality, visual output, and performance of Hybrid-DIA MS technology. The application analyzes DIA mass spectra in real-time to detect peptide transitions of interest. These precursor peptides are then targeted for quantitation on the fly. Targeted scans are fully customizable for resolution, injection time, and ion accumulation to maximize sensitivity.

Moonshot Pro combines various targeted methodologies with DIA, leveraging a transition mass inclusion list for peptides of interest. By scanning DIA windows for candidate peptides, Moonshot Pro enhances the sensitivity and specificity alongside survey scanning based on the high-resolution accurate precursor mass. Quantitative PRM scans only occur after observation of the correct transitions for a peptide, allowing DIA to remain largely uninhibited to maximize scan frequency and sample coverage. Moonshot Pro performs SureQuant analysis with addition of heavy synthetic peptides to trigger the targeting of endogenous peptide counterparts. Customizing targeted scan settings for sensitivity allows endogenous peptide quantification even when abundances are below detection in DIA. MSx mode simultaneously fragments synthetic and endogenous peptides, enhancing quantification. Additionally, samples labeled with TMTpro18 and synthetic peptides labeled with Super Heavy TMTpro provide a means to multiplex the analysis and quantitation. This dramatically increases sample throughput by quantifying target peptides from multiple samples in a single discovery-based DIA analysis, enhancing data completeness for targets of interest, with potential for use in proteogenomic and translational research studies.