Beyond TurboTMT: Phi-SDM Super-resolution Methods for Next-generation Highly-multiplexed Quantitative Ultrasensitive and Single-Cell Proteomics via TMTPro Complement Ion Deconvolution

Significance of the Topic

Single-cell proteomics reveals cellular heterogeneity critical for understanding biology, disease progression, and therapeutic responses. Achieving high multiplexing with accurate quantitation in low-input samples remains challenging due to limited ion currents and co-isolation artifacts.

Study Objectives and Overview

This work investigates the application of phase-constrained spectrum deconvolution (Phi-SDM) focused on high-mass tandem mass tag complement (TMTc) ions, aiming to:

• Enable ultra-high resolution deconvolution of nearly isobaric 15N/13C TMTc species.
• Improve quantitative accuracy in highly multiplexed single-cell analyses.
• Demonstrate operational workflows on commercial Orbitrap platforms.

Methodology and Instrumentation

• Sample preparation: SCoPE-MS style labeling of single-cell equivalents and carrier material with TMTPro reagents.
• LC-MS systems: Thermo Scientific Q Exactive HF-X with Evosep Plus nanoLC; Orbitrap Exploris 480 with Vanquish Neo UHPLC.
• Acquisition: Transients at nominal 120K and 240K resolution using eFT or Phi-SDM processing; ion fill-times up to 512 ms.
• Data analysis: Xcalibur software for spectrum visualization and deconvolution.

Main Results and Discussion

• Conventional eFT at 240K resolution fails to resolve 12 TMTProC species due to insufficient resolution.
• Phi-SDM on Exploris 480 achieves >600K empirical resolution, fully resolving 12 complement ions at 256 ms and 512 ms transients.
• On Q Exactive HF-X, Phi-SDM processing at 240K settings separated 12 species, eliminating co-isolation artifacts between carrier and single-cell channels.
• Partial peak splitting at 120K indicates the limits of lower transient lengths but confirms improved resolution over eFT.

Benefits and Practical Applications

• Accurate quantitation in multiplexed single-cell proteomics without reliance on MS3 workflows.
• Reduced co-isolation interference enhances sensitivity and quantitation precision.
• Compatibility with existing high-resolution Orbitrap platforms and standard SCoPE-MS workflows.

Future Trends and Potential Applications

• Integration of Phi-SDM across broader mass ranges to further streamline single-cell proteomics.
• Development of automated data pipelines for real-time deconvolution and quantitation.
• Extension to other isobaric labeling strategies and complex biological samples.
• Potential for clinical and pharmaceutical applications requiring ultrasensitive proteomic analysis.

Conclusion

Phi-SDM directed at TMT complement ions significantly enhances resolution and quantitative accuracy in next-generation single-cell proteomics. This approach overcomes limitations of conventional eFT, offering an operational workflow for ultrasensitive, highly multiplexed analyses.

Reference

• Budnik B., Levy E., Harmange G., et al. Mass spectrometry of single mammalian cells quantifies proteome heterogeneity during cell differentiation. Genome Biol. 2018;19:161.
• Specht H., Emmott E., Petelski A., et al. Single-cell proteomic and transcriptomic analysis of macrophage heterogeneity using SCoPE2. Genome Biol. 2021;22:50.
• Petelski A., Emmott E., Leduc A., et al. Multiplexed single-cell proteomics using SCoPE2. Nat. Protoc. (in press) 2021.
• Grinfeld D., Aizikov K., Kreutzmann A., Damoc E., Makarov A. Phase-Constrained Spectrum Deconvolution for Fourier Transform Mass Spectrometry. Anal. Chem. 2017;89(2):1202–1211.
• Kelstrup C.D., Aizikov K., Batth T.S., et al. Limits for Resolving Isobaric Tandem Mass Tag Reporter Ions Using Phi-SDM. J. Proteome Res. 2018;17(11):4008–4016.