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Mass Spectrometric Strategies to Improve the Identification of Pt(II)-Modification Sites on Peptides and Proteins

  • Huilin Li
  • Jonathon R. Snelling
  • Mark P. Barrow
  • James H. Scrivens
  • Peter J. Sadler
  • Peter B. O’ConnorEmail author
Research Article

Abstract

To further explore the binding chemistry of cisplatin (cis-Pt(NH3)2Cl2) to peptides and also establish mass spectrometry (MS) strategies to quickly assign the platinum-binding sites, a series of peptides with potential cisplatin binding sites (Met(S), His(N), Cys(S), disulfide, carboxyl groups of Asp and Glu, and amine groups of Arg and Lys, were reacted with cisplatin, then analyzed by electron capture dissociation (ECD) in a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS). Radical-mediated side-chain losses from the charge-reduced Pt-binding species (such as CH3S or CH3SH from Met, SH from Cys, CO2 from Glu or Asp, and NH2 from amine groups) were found to be characteristic indicators for rapid and unambiguous localization of the Pt-binding sites to certain amino acid residues. The method was then successfully applied to interpret the top-down ECD spectrum of an inter-chain Pt-crosslinked insulin dimer, insulin + Pt(NH3)2 + insulin (>10 kDa). In addition, ion mobility MS shows that Pt binds to multiple sites in Substance P, generating multiple conformers, which can be partially localized by collisionally activated dissociation (CAD). Platinum(II) (Pt(II)) was found to coordinate to amine groups of Arg and Lys, but not to disulfide bonds under the conditions used. The coordination of Pt to Arg or Lys appears to arise from the migration of Pt(II) from Met(S) as shown by monitoring the reaction products at different pH values by ECD. No direct binding of cisplatin to amine groups was observed at pH 3 ~ 10 unless Met residues were present in the sequence, but noncovalent interactions between cisplatin hydrolysis and amination [Pt(NH3)4]2+ products and these peptides were found regardless of pH.

Key words

FTICR Ion mobility Post-translational modifications 

Notes

Acknowledgments

H.L. was supported by the Warwick Postgraduate Research Scholarship (WPRS) and the Departmental Studentship. J.R.S. was supported by the EPSRC/RSC. Financial support from NIH (NIH/NIGMS-R01GM078293), the ERC (247450), the Warwick Centre for Analytical Science (EPSRC funded EP/F034210/1), EPSRC (BP/G006792), and Advantage West Midlands Science City are gratefully acknowledged.

Supplementary material

13361_2014_877_MOESM1_ESM.docx (1.9 mb)
ESM 1 (DOCX 1956 kb)

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Copyright information

© American Society for Mass Spectrometry 2014

Authors and Affiliations

  • Huilin Li
    • 1
  • Jonathon R. Snelling
    • 2
  • Mark P. Barrow
    • 1
  • James H. Scrivens
    • 2
  • Peter J. Sadler
    • 1
  • Peter B. O’Connor
    • 1
    Email author
  1. 1.Department of ChemistryUniversity of WarwickCoventryUK
  2. 2.School of Life ScienceUniversity of WarwickCoventryUK

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