Simultaneous Quantitation and Discovery (SQUAD) metabolomics workflow implementing parallel analysis on Thermo Ascend Tribrid instrument

Significance of the Topic

Simultaneous analysis of known and unknown metabolites accelerates biomarker discovery and quantitative studies while conserving precious biological samples. The SQUAD workflow addresses the need for high-throughput metabolomics by uniting targeted quantitation and untargeted discovery in a single injection, reducing instrument variability and sample consumption.

Purpose and Study Overview

This work describes the development and validation of a single-injection SQUAD metabolomics protocol on the Thermo Scientific™ Ascend™ Tribrid platform. The approach aims to quantify selected isotope-labeled standards accurately and, in parallel, discover and annotate unknown features in NIST SRM 1950 plasma.

Methodology and Instrumentation

Metabolites were extracted from plasma with 80% methanol and separated by reversed-phase HPLC using Hypersil GOLD™ columns. Data acquisition leveraged the Ascend Tribrid system configured for:

• Full-scan, high-resolution Orbitrap MS1 for untargeted discovery.
• Targeted tMS2 quantitation in the linear ion trap for isotope-labeled internal standards.
• Advanced deep-scan AcquireX workflow to maximize MS/MS coverage.

Main Results and Discussion

Quantitative performance of the linear ion trap achieved a limit of quantitation down to 5 femtomoles on column and a dynamic range spanning six orders of magnitude for amino acids such as phenylalanine, isoleucine, leucine, tyrosine, and tryptophan. Comparative data showed:

• 55% more MS1 ions accumulated in Ascend versus legacy Tribrid systems.
• 25% more MS2 spectra acquired, yielding 15% more annotated compounds.
• A strong linear correlation (R2 > 0.998) across 2.5 nM to 2.5 mM for targeted analytes.

High-resolution Orbitrap data combined with enhanced AcquireX fragmentation improved annotation confidence across a broader metabolite range.

Benefits and Practical Applications

The SQUAD workflow offers laboratories a consolidated assay merging targeted quantitation and global profiling. Benefits include reduced analysis time, minimized sample requirement by avoiding multiple injections, and enhanced discovery of low-abundance metabolites alongside precise quantitation of known biomarkers.

Future Trends and Opportunities

Further integration of intelligent acquisition algorithms and machine learning–driven data processing will expand coverage and streamline annotation. Emerging hybrid platforms may incorporate additional ion mobility separation or parallel reaction monitoring to deepen insight into complex biological matrices.

Conclusion

The SQUAD metabolomics approach on the Ascend Tribrid instrument provides a powerful, single-injection solution that couples sensitive, wide-range quantitation with comprehensive discovery. This unified workflow enhances throughput, data quality, and sample efficiency, positioning it as a valuable tool for research and quality-control laboratories engaged in metabolomics studies.

Instrumentation Used

• Thermo Scientific™ Orbitrap Ascend™ Tribrid Mass Spectrometer
• Thermo Scientific™ Hypersil GOLD™ HPLC System
• AcquireX Deep-Scan Workflow
• Thermo Scientific™ TraceFinder™ 5.1 and Compound Discoverer™ 3.3 Software