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Development of novel isobaric tags enables accurate and sensitive multiplexed proteomics using complementary ions

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Abstract

High-throughput quantitative analysis of the cells’ proteomes across multiple conditions such as various perturbations and different time points is essential for gaining insights into treatment-induced biological responses or disease pathological states. The advancements in mass spectrometry instrumentation and isobaric labeling methods provided useful tools to help address such demands. However, the current widely adopted isobaric labeling methods such as tandem mass tag (TMT) and isobaric tags for relative and absolute quantitation (iTRAQ) are based on low-mass reporter ions, which are indistinguishable among different peptide analytes, to achieve relative quantification. Therefore, these methods intrinsically suffer from severe ratio distortion when analyzing complex samples due to peptide coelution and cofragmentation. Here, we developed a novel set of isobaric tags named dimethylated leucine complementary ion (DiLeuC) and relied on complementary ions for relative quantification, in which the complementary ions are the remanent peptide segments after fragmentation in the high-mass range. Since those residual peptide fragments are precursor-specific, they retain the relative abundance information in an interference-free manner even in a complex matrix environment. The quantification accuracy of our method was validated in a two-proteome model where the yeast proteome was spiked with a strong background human proteome as interference. In addition, we also applied this strategy to single-cell proteome analysis, demonstrating its potential utility for sensitive high-throughput quantitative proteomics.

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Acknowledgements

The authors wish to thank SCIENION US Inc. for providing access to a cellenONE system for some of the single-cell analysis work.

Funding

This study was supported in part by grant funding from the NIH (R01AG052324, R01DK071801, and P41GM108538). L.L. acknowledges funding support of NIH funding R01AG078794 and shared instrument grants (NIH-NCRR S10RR029531, S10OD028473, and S10OD025084), a Vilas Distinguished Achievement Professorship, and the Charles Melbourne Johnson Distinguished Chair Professorship with funding provided by the Wisconsin Alumni Research Foundation and the University of Wisconsin–Madison School of Pharmacy.

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Author notes

  1. Miyang Li and Min Ma contributed equally to this work.

Authors and Affiliations

  1. Department of Chemistry, University of WI–Madison, Madison, WI, 53706, USA

    Miyang Li & Lingjun Li

  2. School of Pharmacy, University of WI–Madison, 777 Highland Avenue, Madison, WI, 53705, USA

    Min Ma & Lingjun Li

  3. Lachman Institute for Pharmaceutical Development, School of Pharmacy, University of WI–Madison, Madison, WI, 53705, USA

    Lingjun Li

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  1. Miyang Li
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Correspondence to Lingjun Li.

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Published in the topical collection Recent Advances in Ultrasensitive Omics Techniques with guest editor Joseph Zaia.

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Li, M., Ma, M. & Li, L. Development of novel isobaric tags enables accurate and sensitive multiplexed proteomics using complementary ions. Anal Bioanal Chem 415, 6951–6960 (2023). https://doi.org/10.1007/s00216-023-04877-3

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  • DOI: https://doi.org/10.1007/s00216-023-04877-3

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