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Analysis of protein mixtures from whole-cell extracts by single-run nanoLC-MS/MS using ultralong gradients

Abstract

The majority of proteome-wide studies rely on the high separation power of two-dimensional liquid chromatography–tandem mass spectrometry (2D LC-MS/MS), often combined with protein prefractionation. Alternative approaches would be advantageous in order to reduce the analysis time and the amount of sample required. On the basis of the recent advances in chromatographic and mass spectrometric instrumentation, thousands of proteins can be identified in a single-run LC-MS/MS experiment using ultralong gradients. Consequently, the analysis of simple proteomes or clinical samples in adequate depth becomes possible by performing single-run LC-MS/MS experiments. Here we present a generally applicable protocol for protein analysis from unseparated whole-cell extracts and discuss its potential and limitations. Demonstrating the practical applicability of the method, we identified 2,761 proteins from a HeLa cell lysate, requiring around 10 h of nanoLC-MS/MS measurement time.

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Figure 1: The flow paths of the nanoLC system.
Figure 2: The UV chromatogram of a QC sample is shown together with the pressure curve of the nanoLC separation.
Figure 3: Triplicates of a CID-based LC-MS/MS analysis of 1-μg HeLa lysate using varying gradient times.
Figure 4: LC-MS/MS analysis of varying amounts of HeLa lysate using HCD for fragmentation.

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Acknowledgements

This work was funded by Boehringer Ingelheim, the Christian Doppler Research Association, the Austrian Proteomics Platform within the Austrian GenomeResearch program (GEN-AU), the Austrian Science Fund via the Special Research Program Chromosome Dynamics (SFB-F3402) and the European Commission via the FP7 projects MeioSys and Prime XS. The technical support of the other members of the Mechtler group, especially of G. Krssakova, is gratefully acknowledged.

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Contributions

T.K. and K.M. designed the study. T.K. and P.P. performed experiments and analyzed the data. R.S. provided conceptual input and assisted in the experimental design. T.K. and K.M. supervised the project. All authors discussed the experimental results. T.K. wrote the manuscript.

Corresponding authors

Correspondence to Thomas Köcher or Karl Mechtler.

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The authors declare no competing financial interests.

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Köcher, T., Pichler, P., Swart, R. et al. Analysis of protein mixtures from whole-cell extracts by single-run nanoLC-MS/MS using ultralong gradients. Nat Protoc 7, 882–890 (2012). https://doi.org/10.1038/nprot.2012.036

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