A TMTpro 18plex Proteomics Standard for Assessing Protein Measurement Accuracy and Precision

Significance of the Topic

The development of a TMTpro 18plex yeast digest standard addresses critical challenges in multiplexed proteomics by providing a tool to evaluate and optimize quantification accuracy and precision across diverse LC–MS acquisition strategies.

Objectives and Study Overview

• Design and validate a prototype TMTpro 18plex standard using yeast strains with defined knockout proteins.
• Compare the performance of high-resolution MS2, SPS-MS3 and real-time search (RTS) MS3 acquisition modes.
• Assess the impact of FAIMS separation on measurement fidelity.

Used Instrumentation

• Thermo Scientific Orbitrap Eclipse Tribrid mass spectrometer
• Thermo Scientific Exploris 480 mass spectrometer
• FAIMS Pro interface
• Thermo Scientific EASY-Spray C18 50 cm column
• Thermo Scientific UltiMate 3000 RSLCnano UHPLC system
• Thermo Scientific EASY-nLC 1200 HPLC system

Methodology

• Sample Preparation: A parental Saccharomyces cerevisiae strain (BY4741) and three knockout lines lacking Met6, His4 or Ura2 were grown to OD600 ≈ 3.0. Cells were lysed by bead beating, proteins reduced, alkylated and digested with LysC and trypsin, followed by C18 SPE cleanup.
• TMTpro Labeling: Peptides from each knockout strain were labeled in triplicate and parental peptides in duplicate with distinct TMTpro 18plex tags. Samples were mixed at fixed ratios (100%, 50%, 25%, 12.5%, 6.25% and 0%).
• LC Separation: Peptides were separated on a 50 cm EASY-Spray C18 column using gradients (4–28% or 8–32% acetonitrile with 0.1% formic acid) over 50 or 120 minutes.
• Acquisition Methods: Data were acquired on Orbitrap Eclipse and Exploris 480 instruments using three strategies:
  • High-resolution MS2 (hrMS2)
  • Synchronous precursor selection MS3 (SPS-MS3)
  • Real-time search MS3 (RTS-MS3) with varied close-out thresholds.
  FAIMS Pro was applied in selected experiments to reduce co-isolation interference.
• Data Analysis: Raw files processed in Proteome Discoverer 2.5/3.0 with SEQUEST HT or COMET search engines, 1% FDR, 10 ppm MS1 and 0.02/0.6 Da MS2 tolerance, and co-isolation filters of 50% (MS2) or 70% (MS3).

Results and Discussion

• SPS-MS3 methods significantly improved quantification precision relative to hrMS2 by mitigating ratio compression from co-isolated peptides.
• Inclusion of FAIMS Pro further enhanced measurement accuracy for both MS2 and MS3 workflows by reducing background interference.
• RTS-MS3 acquisition achieved the highest proteome coverage, with a 26% increase in quantified protein groups over SPS-MS3 due to real-time peptide identification and targeted MS3 scans.
• Standard curves for Met6, His4 and Ura2 displayed linearity >0.98 across four highest dilutions, with slight decrease at the lowest concentration; RTS-MS3 reduced background in knockout channels to undetectable levels.

Benefits and Practical Applications

• The TMTpro 18plex yeast standard provides a reproducible benchmark for optimizing multiplex proteomic workflows and instrument performance.
• Advanced acquisition schemes such as RTS-MS3 enable deeper proteome coverage and more accurate quantification, supporting rigorous QA/QC in research and industrial laboratories.

Future Trends and Potential Applications

• Integration of real-time search strategies with machine-learning–driven decision making may further enhance dynamic acquisition efficiency.
• Adoption of multiplexed standards in clinical and large-cohort studies will improve cross-laboratory data comparability and reliability.
• Extension of standardized mixtures to other organisms or cell types and higher-plex reagents will broaden applicability in diverse proteomic investigations.

Conclusion

The TMTpro 18plex yeast digest standard demonstrates a versatile and robust approach to assess and refine multiplexed proteomic assays. SPS-MS3 and particularly RTS-MS3 workflows deliver superior accuracy, precision and proteome depth, facilitating optimized experimental designs and reliable quantitative results.

Reference

1. Paulo JA, O’Connell JD, Gygi SP. A triple knockout (TKO) proteomics standard for diagnosing ion interference in isobaric labeling experiments. Journal of the American Society for Mass Spectrometry. 2016;27(10):1620–1625.