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Drift time-specific collision energies enable deep-coverage data-independent acquisition proteomics


We present a data-independent acquisition mass spectrometry method, ultradefinition (UD) MSE. This approach utilizes ion mobility drift time-specific collision-energy profiles to enhance precursor fragmentation efficiency over current MSE and high-definition (HD) MSE data-independent acquisition techniques. UDMSE provided high reproducibility and substantially improved proteome coverage of the HeLa cell proteome compared to previous implementations of MSE, and it also outperformed a state-of-the-art data-dependent acquisition workflow. Additionally, we report a software tool, ISOQuant, for processing label-free quantitative UDMSE data.

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Figure 1: UDMSE concept.
Figure 2: ISOQuant improves data quality and reproducibility.
Figure 3: Comparison of MSE, HDMSE and UDMSE.


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We thank R. Spohrer for excellent sample preparation; W. Thompson and K. Giles for discussions on ion-mobility separations and data-independent acquisition methods; A. Savidor and A. Gabashvili for assistance in the DDA analysis; all ISOQuant beta testers for their continuing critical evaluation of the software; and H. Vissers and K. Richardson for discussions on data evaluation. This work was supported by grants from the Deutsche Forschungsgemeinschaft (INST 371/23-1 FUGG) to S.T., H.S. and the BMBF (e:Bio Express2Present, 0316179C) to S.T., as well by as the Forschungszentrum Immunologie (FZI), the Naturwissenschaftlich-Medizinische Forschungszentrum (NMFZ) and the Focus Program Translational Neurosciences (FTN) of the Johannes Gutenberg University Mainz.

Author information

Authors and Affiliations



U.D. and S.T. designed and performed the DIA experiments. Y.L. performed the DDA experiments and DDA data evaluation. J.K. designed and wrote the software. J.K. and S.T. developed algorithms. U.D., J.K., P.N. and S.T. analyzed the DIA data. U.D., J.K., H.S. and S.T. contributed to overall design of the project. All authors prepared and reviewed the manuscript.

Corresponding author

Correspondence to Stefan Tenzer.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–13, Supplementary Tables 1–4 and 9, and Supplementary Notes 1 and 2 (PDF 5277 kb)

Supplementary Table 5

List of identified proteins in tryptic HeLa cell lysate after analysis with MSE, HDMSE and UDMSE. (XLSX 4155 kb)

Supplementary Table 6

MaxQuant report of the DDA-analysis of tryptic HeLa cell lysate. (XLSX 4175 kb)

Supplementary Table 7

ISOQuant quantification Excel report for hybrid proteome sample using high confidence settings. (XLSX 1820 kb)

Supplementary Table 8

High confidence and MaxID settings for ISOQuant processing. (XLSX 11 kb)

Supplementary Software

Supplementary Software (ZIP 30644 kb)

Source data

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Distler, U., Kuharev, J., Navarro, P. et al. Drift time-specific collision energies enable deep-coverage data-independent acquisition proteomics. Nat Methods 11, 167–170 (2014).

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