Analytical and Bioanalytical Chemistry

, Volume 411, Issue 24, pp 6275–6285 | Cite as

Detection and fragmentation of doubly charged peptide ions in MALDI-Q-TOF-MS by ion mobility spectrometry for improved protein identification

  • Jens SproßEmail author
  • Alexander Muck
  • Harald GrögerEmail author
Research Paper
Part of the following topical collections:
  1. Close-Up of Current Developments in Ion Mobility Spectrometry


Today, bottom-up protein identification in MALDI-MS is based on employing singly charged peptide ions, which are predominantly formed in the ionization process. However, peptide mass fingerprinting (PMF) with subsequent tandem MS confirmation using these peptide ions is often hampered due to the lower quality of fragment ion mass spectra caused by the higher collision energy necessary for fragmenting singly protonated peptides. Accordingly, peptide ions of higher charge states would be of high interest for analytical purposes, but they are usually not detected in MALDI-MS experiments as they overlap with singly charged matrix clusters and peptide ions. However, when utilizing ion mobility spectrometry (IMS), doubly charged peptide ions can be actively used by separating them from the singly protonated peptides, visualized, and selectively targeted for tandem MS experiments. The generated peptide fragment ion spectra can be used for a more confident protein identification using PMF with tandem MS confirmation, as most doubly protonated peptide ions yield fragment ion mass spectra of higher quality compared to tandem mass spectra of the corresponding singly protonated precursor ions. Mascot protein scores can be increased by approximately 50% when using tandem mass spectra of doubly charged peptide ions, with ion scores up to six times higher compared with ion scores of tandem mass spectra from singly charged precursors.


Doubly charged peptide ions Ion mobility spectrometry (IMS) MALDI-Q-TOF mass spectrometry Protein analysis Proteomics 



The authors thank T. Winkler for the preparation of ene reductase.

Funding information

J.S. and H.G. received generous support from the German Research Foundation (DFG; grant number: INST 215/484-1 FUGG).

Compliance with ethical standards

Conflict of interest

Jens Sproß and Harald Gröger declare no conflict of interest. Alexander Muck is an employee of Waters Corp.

Supplementary material

216_2019_1578_MOESM1_ESM.pdf (2 mb)
ESM 1 (PDF 2021 kb)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Industrial Organic Chemistry and Biotechnology, Faculty of ChemistryBielefeld UniversityBielefeldGermany
  2. 2.Waters GmbHEschbornGermany

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