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

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.

Mass spectrometry based strategy for studies of binding sites and structural changes of cisplatin binding to myoglobin

“…The samples were diluted to 5 μM with 20% methanol/0.1% formic acid immediately before MS analysis. Cisplatin, a moderately toxic compound, should be handled carefully in a safe environment, and the material safety data sheets should be studied carefully …”

Mass spectrometry as a powerful tool to study therapeutic metallodrugs speciation mechanisms: Current frontiers and perspectives

“…[78,79] A combination of different fragmentation techniques (CID and ECD) as well as ion mobility mass spectrometry (IM-MS) allowed the determination of the sulfur atom of the methionine residue of substance P as the favoured binding site of cisplatin, whereas the His residues of angiotensin II and bombesin appeared to be preferred. [78][79][80] Tandem mass spectrometry techniques have been used to characterize cisplatin interactions with were recorded and showed that, even though the interactions between the complexes and the DNA strands were non-covalent in nature, they were strong enough to be observed in the gas phase during the analysis, and confirmed that PPCs do significantly stabilise the duplex structure of the oligonucleotide strands.…”

The emerging role of native mass spectrometry in characterizing the structure and dynamics of macromolecular complexes

“…83 Both gas-phase dissociative approaches were utilized to study metallodrug-peptide complexes and characterize the amino acid binding sites. 84,85 CID, ETD, and higher-energy C-trap dissociation (HCD) were also applied to study a metallodrug-protein complex (i.e., the oxaliplatin-ubiquitin assembly). 86 Among the three approaches, ETD was the most useful for identifying the metal binding sites.…”

“…CID and ECD were also used to characterize nanoscale bilayers (nanodiscs) formed by the binding of phospholipids to a scaffold protein . Both gas‐phase dissociative approaches were utilized to study metallodrug‐peptide complexes and characterize the amino acid binding sites . CID, ETD, and higher‐energy C‐trap dissociation (HCD) were also applied to study a metallodrug‐protein complex (i.e., the oxaliplatin‐ubiquitin assembly) .…”