Thermo Scientific Q Exactive Plus Orbitrap LC-MS/MS System

Importance of the Topic

The integration of characterization, quantification, and confirmation on a single high-resolution mass spectrometer streamlines workflows in pharmaceutical development, proteomics research, and quality control. Reliable detection of low-abundance analytes, intact protein analysis, and confident data interpretation are critical to advancing drug discovery, biotherapeutic characterization, and regulatory compliance.

Objectives and Overview of the Study

This application note introduces the Thermo Scientific Q Exactive Plus hybrid quadrupole-Orbitrap mass spectrometer and its Quanfirmation Plus capabilities. Key goals include:

• Enhancing sensitivity and selectivity for protein identification in complex matrices
• Enabling precise targeted quantitation with high-resolution accurate-mass data
• Expanding intact protein analysis for biotherapeutics with optional modes
• Demonstrating improved throughput and confidence in data-independent acquisition

Methodology and Instrumentation Used

The system combines:

• Quadrupole mass filter with Advanced Quadrupole Technology (AQT) for refined precursor selection and transmission
• Orbitrap mass analyzer delivering high-resolution accurate-mass measurements
• Advanced Active Beam Guide (AABG) to reduce background noise and extend maintenance intervals
• HCD collision cell for robust fragmentation
• Optional intact protein mode and enhanced resolution for top-down proteomics
• Targeted workflows: Parallel Reaction Monitoring (PRM) and High-Resolution/Accurate-Mass Selected Ion Monitoring (HR/AM-SIM)
• Data-Independent Acquisition (DIA) methods processed with Thermo Scientific PinPoint software

Main Results and Discussion

Protein Identification

• Advanced optics and quadrupole improvements led to up to 1.5-fold gains in low-abundance protein identification compared to prior models
• Reliable performance across a range of peptide digest loads (0.1–1000 ng on column)

Intact Protein Analysis

• Intact Protein Mode combined with enhanced resolution achieved isotopic resolution of subunits up to 50 kDa
• Four-fold increase in signal for heavy chain glycoforms of monoclonal antibodies at 140 000 resolution

Targeted Quantitation

• AQT improvements provided up to six-fold transmission gain for small molecules and peptides over a wide isolation width range
• PRM and HR/AM-SIM workflows delivered sensitive quantitation with full qualitative confirmation

Data-Independent Acquisition

• Optimized DIA windows (10–50 amu) yielded consistent precursor isolation and comprehensive protein coverage
• PinPoint software integration enhanced reproducibility and throughput without prior sample-specific method development

Benefits and Practical Applications

The Q Exactive Plus platform offers:

• Single-instrument workflow for qualitative and quantitative assays, reducing time and resource requirements
• High confidence in analyte identity and abundance for DMPK screening, biotherapeutic characterization, and QA/QC
• Flexibility to switch between bottom-up proteomics, intact protein analysis, and targeted assays

Future Trends and Potential Applications

Emerging directions include:

• Further integration of top-down proteomics for characterization of complex protein assemblies
• Automation and machine-learning-driven data analysis to accelerate decision-making
• Expansion into clinical and regulatory workflows requiring validated high-resolution quantitation
• Advancements in ion optics and detectors to push limits of sensitivity and mass range

Conclusion

The Q Exactive Plus mass spectrometer elevates the concept of Quanfirmation by combining advanced quadrupole selection, Orbitrap resolution, and flexible acquisition modes. It delivers robust protein identification, sensitive targeted quantitation, and confident intact protein characterization within a unified platform, addressing the growing demands of pharmaceutical and proteomic laboratories.

Instrumentation

• Thermo Scientific Q Exactive Plus hybrid quadrupole-Orbitrap mass spectrometer
• Advanced Active Beam Guide (AABG)
• Advanced Quadrupole Technology (AQT)
• HCD collision cell
• Optional intact protein mode and enhanced resolution package