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Label-free quantification in ion mobility–enhanced data-independent acquisition proteomics

Nature Protocols volume 11, pages 795–812 (2016)Cite this article

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Abstract

Unbiased data-independent acquisition (DIA) strategies have gained increased popularity in the field of quantitative proteomics. The integration of ion mobility separation (IMS) into DIA workflows provides an additional dimension of separation to liquid chromatography-mass spectrometry (LC-MS), and it increases the achievable analytical depth of DIA approaches. Here we provide a detailed protocol for a label-free quantitative proteomics workflow based on ion mobility–enhanced DIA, which synchronizes precursor ion drift times with collision energies to improve precursor fragmentation efficiency. The protocol comprises a detailed description of all major steps including instrument setup, filter-aided sample preparation, LC-IMS-MS analysis and data processing. Our protocol can handle proteome samples of any complexity, and it enables a highly reproducible and accurate precursor intensity–based label-free quantification of up to 5,600 proteins across multiple runs in complete cellular lysates. Depending on the number of samples to be analyzed, the protocol takes a minimum of 3 d to complete from proteolytic digestion to data evaluation.

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Figure 1: Schematic overview of the protocol.
Figure 2: LC and nanospray performance.
Figure 3: IMS tuning.
Figure 4: Results of HeLa analysis.
Figure 5: Label-free quantification using ISOQuant.

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Acknowledgements

This work was supported by grants from the German Federal Ministry for Education and Research (BMBF; Express2Present, 0316179C), the Deutsche Forschungsgemeinschaft (DFG; TE599/1-1), Mainz University (Research Center for Immunotherapy (FZI), and Focus Program Translational Neurosciences (FTN).

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  1. Ute Distler and Jörg Kuharev: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute for Immunology, University Medical Center of the Johannes-Gutenberg University Mainz, Mainz, Germany

    Ute Distler, Jörg Kuharev, Pedro Navarro & Stefan Tenzer

  2. Focus Program Translational Neuroscience (FTN), University Medical Center of the Johannes-Gutenberg University Mainz, Mainz, Germany

    Ute Distler

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  1. Ute Distler
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Contributions

U.D., J.K. and S.T. developed the protocol and conducted the experiments. J.K. developed software. U.D., J.K., P.N. and S.T. interpreted the data and drafted the manuscript.

Corresponding author

Correspondence to Stefan Tenzer.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Comparison of sample preparation protocols and LC setup.

In a recent study, Geromanos et al.20 applied an IMS-enhanced MSE-workflow for proteomics using the same instrumentation as in the present protocol. We compared the in-solution digestion as performed by Geromanos et al. (red bars) to the FASP protocol applied in the present study (blue bars) using HeLa cells. LC-MS analysis using MSE and HDMSE acquisition modes were performed as recently described21. Compared to the in-solution digest the number of identified proteins shows an increase of about 16.2% in MSE and 28.4% in HDMSE using FASP digest. Further on, we performed a comparison between the chromatographic setup used in the present protocol and the one used by Geromanos et al. Following parameters were different between the two setups: i) column material (HSS-T3 vs. BEH-C18), ii) column length (25 cm vs. 20 cm), iii) operating temperature (55°C vs. 35°C). Optimizing the chromatographic setup by using HSS-T3 columns (25 cm) and higher separation temperature led to an increase in protein identifications of 33.8% (MSE) and 15.4% (HDMSE), respectively.

Supplementary Figure 2 Chromatographic resolution.

Screenshots of extracted ion chromatograms (XICs) of m/z 534.28 (15 mDa extraction window) are shown over a time frame of 50 min (upper panel) and 5 min (lower panel), respectively. Runs are visualized with the vendor instrument control software MassLynx (version 4.1; Waters Corporation). With the chromatographic setup described in the present protocol peak widths at half height of about 0.1 min at the start of the gradient and about 0.25 min at the end of the gradient can be achieved. The data are also depicted in Figure 2a in the main manuscript.

Supplementary Figure 3 Spray stability.

Screenshots of LC-MS runs performed with stable and instable nanospray conditions. Runs are displayed in vendor instrument control software MassLynx (version 4.1; Waters Corporation). The data from the upper two runs are also depicted in Figure 2b in the main manuscript. Only the uppermost run shows stable nanospray. The other runs were all performed with unstable, sputtering nanospray hampering proper sample analysis. When setting up your system make sure to operate with stable nanospray conditions.

Supplementary Figure 4 Increase in fragmentation efficiency using drift time specific collision energies.

Screenshots of total ion currents (TIC) of LC-MS runs performed with (a) UDMSE and (b) HDMSE. Runs are displayed in vendor instrument control software MassLynx (version 4.1; Waters Corporation). While displaying similar TICs in the MS1 trace, MS2 traces display about 1.5 times higher TICs for UDMSE methods. This observation can be used as a quick initial check if fragmentation efficiencies are increased in your UDMSE method as compared to your HDMSE method.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4, Supplementary Methods 2–4 and Supplementary Manual (PDF 5694 kb)

Supplementary Method 1

MS tune page file. (TXT 85 kb)

Supplementary Table 1

Settings for ISOQuant post-processing for label-free quantification. (XLSX 11 kb)

Supplementary Data 1

ISOQuant protein quantification report for Pierce HeLa sample. (XLSX 1904 kb)

Supplementary Data 2

ISOQuant protein quantification report for FASP HeLa sample. (XLSX 1949 kb)

Supplementary Data 3

Numbers of identified peptides and proteins in HeLa samples. (XLSX 35 kb)

Supplementary Data 4

ISOQuant protein quantification report for hybrid proteome samples acquired with MSE. (XLSX 484 kb)

Supplementary Data 5

ISOQuant protein quantification report for hybrid proteome samples acquired with UDMSE. (XLSX 1087 kb)

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Distler, U., Kuharev, J., Navarro, P. et al. Label-free quantification in ion mobility–enhanced data-independent acquisition proteomics. Nat Protoc 11, 795–812 (2016). https://doi.org/10.1038/nprot.2016.042

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