Targeted Quantitative Proteomics

Importance of the topic

Targeted quantitative proteomics is a critical tool in modern analytical chemistry, enabling precise measurement of selected proteins in complex biological samples. It supports biomarker validation, signaling pathway mapping, and quality control in pharmaceutical and clinical research. High-resolution, accurate-mass (HR/AM) mass spectrometry addresses limitations of traditional selected-reaction monitoring (SRM), offering enhanced selectivity, sensitivity, and throughput.

Objectives and overview of the study

This whitepaper introduces the application of HR/AM mass spectrometry, specifically the Thermo Scientific Q Exactive platform, for targeted protein quantification. It compares conventional SRM on triple quadrupoles with parallel-reaction monitoring (PRM), selected-ion monitoring (SIM), and data-independent acquisition (DIA) workflows. The focus is on demonstrating improved performance in complex matrices, assay flexibility, and streamlined method development.

Methodology and instrumentation

This work leverages Orbitrap-based HR/AM detection for MS and MS/MS analysis. Key methodologies include:

• PRM: Quadrupole isolation followed by high-energy collisional dissociation (HCD) and Orbitrap detection of fragment ions within ±5 ppm windows.
• SIM: Targeted precursor isolation without fragmentation, using narrow mass windows at up to 140,000 resolving power.
• DIA: Sequential acquisition of MS/MS spectra over wide isolation windows (<25 m/z), with spectral library matching for peptide identification.
• Multiplexed variants (msxSIM, msxDIA): Random or scheduled selection of multiple narrow windows per cycle to boost throughput and reduce interferences.

Instrumentally, the Q Exactive MS provides:

• Up to 140,000 resolving power.
• Mass accuracy <1 ppm.
• Up to 12 Hz HCD MS/MS at 17.5k resolution.
• Parallel ion filling and detection with spectrum multiplexing.

Main results and discussion

HR/AM PRM assays achieved attomole-level detection of peptides spiked into E. coli and CSF digests, eliminating background interference common in SRM. SIM experiments enriched precursor ions 5–50× over full‐scan MS, enabling detection down to low amol levels with signal‐to‐noise ratios >40. DIA provided comprehensive MS/MS coverage for mid- to high‐abundance proteins, while msxDIA improved selectivity by demultiplexing narrow windows. All methods demonstrated linear quantification over four to five orders of magnitude.

Benefits and practical applications

• High selectivity: Resolves near-isobaric ions by tens of ppm.
• High sensitivity: Detects low-abundance peptides in complex matrices.
• Broad dynamic range: Accurate quantification from amol to fmol levels.
• Method flexibility: Supports PRM, SIM, DIA, and multiplexed workflows without extensive assay optimization.
• Streamlined development: Eliminates manual transition selection; leverages software for automated method creation.

Future trends and applications

Continued advances will focus on deeper multiplexing, real-time acquisition steering, and integration with ion mobility separations. Artificial intelligence–driven data processing and cloud-based spectral libraries will enable retrospective mining of large DIA datasets. Further miniaturization and high-throughput LC coupling will expand applications in clinical diagnostics and bioprocess monitoring.

Conclusion

High-resolution, accurate-mass mass spectrometry on hybrid quadrupole-Orbitrap platforms ushers in a new era of targeted proteomics. By overcoming SRM constraints, HR/AM workflows deliver exceptional selectivity, sensitivity, and productivity, making them indispensable for modern biological and pharmaceutical research.

Instrumental setup

• Thermo Scientific Q Exactive hybrid quadrupole-Orbitrap mass spectrometer
• Thermo Scientific Dionex UltiMate 3000 RSLCnano UHPLC system
• Thermo Scientific EASY-nLC 1000 nano-LC system with EASY-Spray source
• Isotopically labeled peptide standards and retention time calibration mixtures

References

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2. Gallien S, Duriez E, Crone C, Kellmann M, Moehring T, Domon B. Targeted Proteomic Quantification on Quadrupole-Orbitrap Mass Spectrometer. Mol Cell Proteomics. 2012.
3. Gillet LC, Navarro P, Tate S, et al. Targeted data extraction of the MS/MS spectra generated by data independent acquisition: a new concept for consistent and accurate proteome analysis. Mol Cell Proteomics. 2012.
4. Egertson JD, Kuehn A, Merrihew G, et al. Multiplexed Data Independent Acquisition for Comparative Proteomics. ASMS poster. 2012.
5. MacLean B, Tomazela DM, Chambers M, et al. Skyline: an open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics. 2010;26(7):966–968.