Expanding plasma metabolome coverage with enhanced dynamic range and iterative MS/MS acquisition on Orbitrap Excedion mass spectrometer

Significance of the topic

Plasma metabolomics faces an extreme concentration dynamic range where a few highly abundant species dominate ion accumulation, limiting detection and confident MS/MS annotation of lower-abundance metabolites. Enhanced dynamic range (eDR) acquisition aims to rebalance ion filling across the m/z range to boost signal-to-noise (S/N) for medium- and low-abundance ions without sacrificing broad spectral coverage. Improving usable feature detection and high-confidence library matches directly benefits biomarker discovery, exposomics, and large-cohort untargeted studies where depth and reproducibility matter.

Objectives and study overview

This study evaluated how eDR full-scan acquisition on the Orbitrap Excedion mass spectrometer, combined with AcquireX iterative data-dependent acquisition (DDA), affects plasma metabolome coverage and high-confidence MS/MS annotation. Key aims were to (1) compare eDR versus conventional full-scan across multiple plasma-equivalent loads, (2) quantify changes in quality-filtered MS1 compound entities and strict mzCloud MS/MS matches, and (3) test the impact of MS2 isolation width on precursor selectivity and library match quality.

Methodology

Samples and sample prep:

• NIST SRM 1950 human plasma extracts prepared by methanol protein precipitation, centrifugation, evaporation, and reconstitution.
• Multiple extract dilutions and injection volumes were normalized to plasma-equivalent volumes loaded on column (representative loads: 0.58, 1.44, 4.33 µL equivalents).

Chromatography and acquisition:

• UHPLC: Vanquish Horizon system with Hypersil GOLD VANQUISH C18 column (150 × 2.1 mm, 1.9 µm).
• Mass spectrometer: Thermo Scientific Orbitrap Excedion.
• MS1 full-scan: m/z 67–1000 at 120k resolving power; eDR segmented the MS1 range into 16 windows to redistribute ion injection time across the mass range.
• MS/MS: AcquireX-guided iterative Deep Scan DDA with stepped normalized HCD energies (10, 35, 60%) and variable isolation widths (0.4–2.0 Da).
• Representative AcquireX sequences included blank-derived exclusion lists, one MS1 reference injection, two MS1 replicate injections, and 6–7 iterative identification injections.

Data processing and quality filters:

• Raw data processed using Thermo Scientific Compound Discoverer 3.5 and FreeStyle 1.8 SP2.
• MS1 quality filters: coefficient of variation (CV) <20%, formula assignment present, and peak rating >6 in three MS1 reference replicates.
• Strict mzCloud MS/MS filters: CV <20%, non-blank formula, MS1 peak rating ≥6 in 3/3 references, mzCloud match score ≥60, and MS2 spectral purity >60.

Instrumental details (Used instrumentation)

• UHPLC: Thermo Scientific Vanquish Horizon.
• Column: Hypersil GOLD VANQUISH C18, 150 × 2.1 mm, 1.9 µm.
• Mass spectrometer: Thermo Scientific Orbitrap Excedion with eDR capability (MS1 segmented into 16 windows).
• Software: AcquireX for iterative DDA, Compound Discoverer 3.5 and FreeStyle 1.8 SP2 for data processing.

Key results and discussion

MS1 coverage and annotation depth:

• eDR enhanced the number of quality-filtered MS1 compound entities at every tested plasma-equivalent load. At the highest load (4.33 µL equivalent), eDR produced 6,619 quality-filtered MS1 entities versus 1,591 for conventional full scan, a +316% increase.
• eDR gains were largest in absolute and relative terms at the highest sample load, consistent with better handling of high ion loads by redistributing injection time to lower-abundance m/z windows.

MS/MS library matches and spectral quality:

• Strict mzCloud MS/MS matches also increased with eDR. At the highest load, eDR yielded 859 strict mzCloud matches vs 509 with conventional acquisition (+69%).
• Reducing MS2 isolation width (e.g., from 2.0 to 0.4 Da) improved precursor selectivity and reduced co-isolation, increasing MS2 spectral purity and the number of quality-filtered mzCloud matches for precursors below m/z 400.
• Example: paracetamol MS2 purity improved from 45 to 95 when using 0.4 Da isolation, producing cleaner fragments and higher-quality library annotations.

Mechanistic insight (ion injection time redistribution):

• Representative metabolite traces such as cortisone showed that eDR increases S/N primarily by increasing ion injection time for less abundant ions; the largest injection-time gains were seen at lower abundance levels. At higher local abundance, less IT was needed, indicating time was shifted toward weaker signals.
• Numerical examples in the study illustrate multi-fold increases in S/N and IT for lower-abundance features under eDR relative to conventional scan.

Chromatography and practical upper limits:

• Phenylalanine traces monitored across the tested load range (0.58–4.33 µL equivalents) demonstrated stable retention time and peak shape, indicating that chromatographic performance was maintained at higher loads and that the spectrometer, not chromatography, was the primary limiter of usable signal under these conditions.

Benefits and practical applications

• Substantially deeper MS1 feature detection and more high-confidence MS/MS identifications in untargeted plasma metabolomics workflows.
• Improved detection of low- and medium-abundance metabolites without increasing cycle time or losing broad mass-range coverage.
• Enhanced precursor selectivity via narrower MS2 isolation increases annotation confidence, beneficial for small-molecule library matching and downstream biological interpretation.
• Applicable to biomarker discovery, exposome research, population metabolomics, and studies where maximizing identification depth per run is critical.

Limitations and considerations

• eDR performance depends on appropriate parameterization (number of MS1 windows, target IT redistribution) and may require method optimization for different sample types or LC conditions.
• Quadrupole isolation performance constrains the benefit of very narrow MS2 widths for precursors above certain m/z ranges; comparisons in this study were limited to precursors below m/z 400 for narrow-isolation optimization.
• Although eDR improves MS signal balance, the ultimate upper limit of useful loading remains governed by chromatographic integrity and column performance.

Future trends and potential applications

• Integration with ion mobility separation and orthogonal gas-phase fractionation could further enhance depth and deconvolution of coeluting isobars.
• Real-time, feedback-driven acquisition strategies (e.g., machine-learning-guided windowing or adaptive DDA thresholds) could make dynamic redistribution more selective and sample-specific.
• Expansion of high-quality spectral libraries, and automated spectral deconvolution tools, will amplify the practical gains of eDR by converting improved MS2 purity into more identifications.
• Application to other complex matrices (urine, CSF, tissue extracts) and larger cohort studies to test robustness and reproducibility at scale.

Conclusions

Enhanced dynamic range (eDR) acquisition on the Orbitrap Excedion substantially increases quality-filtered MS1 feature counts and strict mzCloud MS/MS matches in NIST SRM 1950 plasma when combined with AcquireX iterative DDA. The principal mechanism is redistribution of ion injection time across segmented MS1 windows, elevating S/N for lower-abundance ions. Narrowing MS2 isolation width further improves MS2 purity and library match rates for low-mass precursors. Together, these optimizations translate to deeper, higher-confidence untargeted plasma metabolomics datasets, with clear utility for discovery research, provided methods are tuned for the sample matrix and chromatographic conditions.

References

• Kaczmarek M, Amer B, Deshpande R, Bird S. Expanding plasma metabolome coverage with enhanced dynamic range and iterative MS/MS acquisition on Orbitrap Excedion mass spectrometer. Thermo Fisher Scientific application note/whitepaper. 2026. (NIST SRM 1950 plasma evaluation; AcquireX iterative DDA; Compound Discoverer 3.5).