High-Throughput Equine Doping Controls on a Trapped Ion Mobility Quadrupole-Time-of-Flight Mass Spectrometer: Technical Considerations of dia/slice/prmPASEF Applied to the Long-Term Detection of Monoclonal Antibodies.

High-throughput screening methods using advanced mass spectrometry techniques were evaluated to detect monoclonal antibodies in horse plasma for doping control, comparing three data acquisition strategies in terms of sensitivity and performance. The study aimed to improve long-term detection capabilities while balancing speed and complexity in analysis.

Background and Objective

• Doping control in equine sports requires sensitive and rapid detection methods for prohibited substances, including monoclonal antibodies.
• Mass spectrometry (MS) coupled with liquid chromatography is widely used for proteomics and doping analyses due to its sensitivity and specificity.
• Recent developments involve data-independent acquisition (DIA) methods leveraging scanning quadrupoles to improve throughput and data quality.
• The study focuses on evaluating three DIA-based acquisition strategies—diaPASEF, slicePASEF, and prmPASEF—on trapped ion mobility spectrometry quadrupole-time-of-flight (TIMS-Q-TOF) MS instruments.
• Objective: To determine which acquisition method best balances sensitivity, throughput, and data complexity for detecting humanized monoclonal antibodies in equine plasma over long periods post-administration.

Overview of Data Acquisition Methods

• diaPASEF: A DIA method that uses a scanning quadrupole combined with ion mobility for untargeted proteomics. It is optimized for complexity and high sample input.
• slicePASEF: An advanced DIA approach that slices the ion mobility space for more focused acquisition, aiming to improve sensitivity and throughput.
• prmPASEF: A parallel reaction monitoring (PRM) technique integrated with ion mobility for targeted, highly sensitive detection.

Experimental Approach

• Horse plasma samples were collected over several weeks following administration of humanized monoclonal antibodies to mimic doping scenarios.
• The three DIA methods were applied using a TIMS-Q-TOF mass spectrometer configured for high-throughput screening.
• Evaluations included sensitivity in antibody detection, data complexity, and suitability for untargeted versus targeted proteomics contexts.

Key Findings

• slicePASEF demonstrated superior performance to diaPASEF for detecting monoclonal antibodies in equine plasma, nearly matching the sensitivity of prmPASEF.
• slicePASEF’s improved sensitivity supports its potential for long-term detection in doping control, which is critical for catching illicit administration weeks after treatment.
• diaPASEF, while less sensitive for monoclonal antibody detection, excels in untargeted proteomics, especially when large amounts of input material are used.
• The high complexity of slicePASEF data and current limitations of data processing algorithms limit its universal application, particularly for untargeted studies.
• prmPASEF remains the most sensitive method but is more targeted and may not be ideal for untargeted screening or where broad analyte coverage is needed.

Technical Considerations and Implications

• Trade-offs exist between sensitivity, throughput, data complexity, and analytical robustness among the methods.
• slicePASEF offers a promising balance for doping control screening, improving sensitivity while maintaining relatively high throughput.
• Ongoing improvements in data processing software are necessary to fully exploit slicePASEF’s complex datasets.
• Selection of methodology should consider the specific goals: broad untargeted proteomics favors diaPASEF, sensitive targeted monitoring favors prmPASEF, and balanced long-term detection benefits from slicePASEF.
• Using TIMS-Q-TOF technology allows enhanced separation and speed, critical for ultra-short liquid chromatography gradients and rapid analyses.

Conclusion

• This study highlights the potential of slicePASEF as a valuable tool for high-throughput equine doping control, with sensitivity close to the targeted prmPASEF method but with greater screening versatility.
• Continued technological and computational improvements will further enhance detection capabilities and support regulatory applications in equine sports.