New Quadrupole-Ion Trap-Orbitrap Mass Spectrometer Combined with Real Time Search Enhances Proteome Coverage and Quantification Accuracy in Multiplexing Workflows

Significance of the Topic

Multiplexed proteomic workflows based on isobaric tags such as TMT revolutionize high-throughput protein quantitation by enabling simultaneous analysis of multiple samples. However, co-isolated peptides and MS2 interference limit quantitative accuracy and depth of coverage. Integrating synchronous precursor selection MS3 (SPS-MS3) with real-time database searching (RTS-MS3) on advanced tribrid platforms addresses these challenges and enhances both sensitivity and precision.

Study Objectives and Overview

This study evaluates the performance of the Thermo Scientific Orbitrap Eclipse Tribrid mass spectrometer with modified hardware and real-time search for TMT-based quantitation. Three sample sets were analyzed: TMT11-plex yeast digest, TMT10-plex labeled 293T cell lines spiked with a six-protein digest, and TMT10-plex depleted human plasma. The RTS-SPS-MS3 workflow was benchmarked against conventional MS2, SPS-MS3, and data-independent acquisition (DIA) methods in terms of proteome coverage, quantification accuracy, and throughput.

Methodology and Instrumentation

• Sample Preparation: Standard Pierce TMT11plex yeast digest; human 293T peptides and depleted plasma peptides labeled with TMT-10plex; six-protein standards spiked at defined ratios.
• Chromatography: Reversed-phase LC separations using single-phase and fractionated workflows with 60–120 min gradients.
• Mass Spectrometry: Orbitrap Eclipse Tribrid quadrupole-ion trap-Orbitrap with real-time search enabled; parameters included 10 ppm MS1 tolerance, 0.6 Da MS2 tolerance, co-isolation threshold at 75%, Xcorr ≥1, ΔCn ≥0.1.
• Data Analysis: Thermo Proteome Discoverer 2.4 for TMT data; DIA-NN for DIA analysis; FDR set at 1% for PSMs, peptides, and proteins.

Key Results and Discussion

• Proteome Coverage: RTS-SPS-MS3 quantified >9 700 protein groups from 12 µg TMT-labeled 293T peptides and >1 400 proteins from plasma within 24 h and 18 h analysis windows, respectively, outperforming MS2 and SPS-MS3 modes.
• Peptide Groups: Over 80 000 peptide groups identified in cell line samples; RTS-SPS-MS3 achieved an interference-free index (IFI) improvement from ~64% (SPS-MS3) to >83% (RTS-SPS-MS3).
• Quantitative Accuracy: RTS-MS3 measured known spike ratios with deviations <10% from expected, reducing interference by ~20% relative to MS2 quantification.
• Comparison to DIA: While RTS-SPS-MS3 offered deeper coverage, DIA achieved marginally higher accuracy in some channels; integration of both strategies can provide orthogonal validation.

Benefits and Practical Applications

• Enhanced Depth: Enables comprehensive profiling of complex proteomes with minimal missing values in multiplex workflows.
• Improved Precision: Real-time search selects optimal MS3 fragments, reducing ratio distortion from co-isolated species.
• Efficiency: Intelligent acquisition maximizes useful MS3 scans, balancing duty cycle and coverage.
• Versatility: Applicable to diverse sample types including cell lysates, plasma, and standard mixtures for biological and clinical research.

Future Trends and Opportunities

Advances in on-the-fly spectral matching and AI-driven acquisition promise further gains in throughput and accuracy. Hybrid approaches combining RTS-MS3 with parallel DIA or novel ion mobility separations may unlock deeper coverage and dynamic range. Continued optimization of instrument control, database algorithms, and multiplex chemistries will expand applications in biomarker discovery, clinical proteomics, and systems biology.

Conclusion

RTS-SPS-MS3 on the Orbitrap Eclipse sets a new benchmark for multiplexed proteomic quantitation, delivering superior coverage and quantification fidelity compared to traditional MS2, SPS-MS3, and DIA strategies. This workflow enables reliable, high-throughput protein analysis essential for complex biological studies.

Instrumentation

• Thermo Scientific Orbitrap Eclipse Tribrid Mass Spectrometer
• Thermo Scientific Pierce TMT11plex Yeast Digest Standard and TMT-10plex Reagents
• Top14 Abundant Protein Depletion Spin Columns
• Reversed-Phase LC System with 60–120 min Gradients
• Thermo Proteome Discoverer 2.4 and DIA-NN Software

References

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