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Ion source-dependent performance of 4-vinylpyridine, iodoacetamide, and N-maleoyl derivatives for the detection of cysteine-containing peptides in complex proteomics

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Abstract

Cysteine is unique among the proteinogenic amino acids due to its ability to form disulfide bonds. While this property is of vital importance for protein structures and biological processes, it causes difficulties for the mass spectrometric identification of cysteine-containing peptides. A common approach to overcome these problems in bottom-up proteomics is the reduction and covalent modification of sulfhydryl groups prior to enzymatic digestion. In this study, established alkylating agents and N-maleoyl amino acids with variable hydrophobicity were characterized with respect to a variety of relevant parameters and subsequently evaluated in a large-scale analysis using different ion sources. Depending on the compound, the ion source had a profound impact on the relative and absolute identification of cysteine-containing peptides. The best results were obtained by derivatization of the cysteine residues with 4-vinylpyridine and subsequent matrix-assisted laser desorption ionization (MALDI). Modification with 4-vinylpyridine increased the number of cysteine-containing peptides identified with any other compound using LC-MALDI/MS at least by a factor of 2. This experimental observation is mirrored by differences in the gas-phase basicities, which were computed for methyl thiolate derivatives of the compounds using density functional theory. With electrospray ionization (ESI), complementary use of reagents from three different compound classes, e.g., iodoacetamide, 4-vinylpyridine, and N-maleoyl beta-alanine, was beneficial compared to the application of a single reagent.

Cysteine-containing peptides are underrepresented in standard large scale proteomic experiments. However, deliberate matching of alkylating agent and ion source can help to increase identification rates, as outlined by characterization and validation of various reagents for cysteine-derivatization.

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Acknowledgments

The authors thank Friedrich Mandel and the team of the Agilent Demolab Waldbronn for their kind support during the Agilent Mass Spec Research Summer 2014. We are most thankful to Prof. Dr. Karl Gademann and to Prof. Dr. Bernd F. Straub for hosting us in their groups. Jaguar licenses were provided by the bwgrid. We gratefully acknowledge the financial support from the German Research Foundation (DFG grant BE 5492/1-1), the German Cancer Research Center, the Dietmar Hopp Stiftung, and the Boehringer Ingelheim Fonds.

Conflict of interest

The authors declare that they have no competing interests.

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Authors and Affiliations

  1. German Cancer Research Center, Im Neuenheimer Feld 280, 69120, Heidelberg, Germany

    Wiebke Nadler, Philipp Walch, Sabrina Hanke, Mathis Baalmann, Alexander Kerner, Andreas Trumpp & Christoph Roesli

  2. Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM gGmbH), Im Neuenheimer Feld 280, 69120, Heidelberg, Germany

    Wiebke Nadler, Philipp Walch, Sabrina Hanke, Alexander Kerner, Andreas Trumpp & Christoph Roesli

  3. Department of Chemistry, University of Zurich, Winterthurerstr 190, 8057, Zurich, Switzerland

    Regina Berg

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  1. Wiebke Nadler
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Correspondence to Christoph Roesli.

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Nadler, W., Berg, R., Walch, P. et al. Ion source-dependent performance of 4-vinylpyridine, iodoacetamide, and N-maleoyl derivatives for the detection of cysteine-containing peptides in complex proteomics. Anal Bioanal Chem 408, 2055–2067 (2016). https://doi.org/10.1007/s00216-015-9113-4

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  • DOI: https://doi.org/10.1007/s00216-015-9113-4

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