Mass spectrometric strategies to improve the identification of Pt(II)-modification sites on peptides and proteins.

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.

Characterization of complex polysorbate formulations by means of shape-selective mass spectrometry.

Complex synthetic formulations based on polysorbates can be challenging to characterize. They may be composed of many similar products including those of the same molecular weight, which cannot be readily separated by separation science approaches. Carbon number variation and ethylene oxide distribution add to the complexity. The properties of these formulations will be dependent on the chemical structure and relative concentration of formulation components. Here we describe the use of two experimental approaches based on mass spectrometry to provide enhanced characterization of these formulations. The first utilizes an atmospheric pressure solids analysis probe to rapidly determine the percentage content of individual esters in a formulation. These are shown to be in good agreement with product specification sheets. In a second approach, mobility separation has been integrated into a MALDI-MS/MS experiment to categorize major, minor, and trace ingredients. Components of identical molecular mass in the polysorbate formulations have been separated by ion mobility and then fragmented for additional characterization. The rapidity and level of structural detail provided by these experiments offers a significant opportunity to develop practical screening methods for complex formulations.

New structural insights into mechanically interlocked polymers revealed by ion mobility mass spectrometry.

Mechanically interlocked polymers can possess significant additional physical properties, in comparison to those associated with their constituent parts. Their unique properties make them attractive for a range of potential applications, such as as biomaterials and molecular machines. Their efficient and reproducible synthesis is therefore of much interest. Both their synthesis and subsequent characterization are intriguing yet demanding. The properties of mechanically interlocked polymeric systems depend not only on the properties of their individual components but also on the topology of the subsequent product. Here traveling wave ion mobility mass spectrometry has been used to investigate the structural properties of a polyrotaxane system. Ion mobility studies reveal that this system remains linear in form with increase in size. Both ion mobility studies and tandem mass spectrometry studies indicate that the macrocycle preferentially remains associated with the ammonium moiety of the polymeric repeat unit and is impeded from moving freely along the axle. This is consistent with NMR observations of the average structure. Analysis of mechanically interlocked polymers by ion mobility mass spectrometry provides additional structural insights into these systems relating to dynamics, heterogeneity, and topology. This molecule-specific information is vital in order to understand the origin of a system's functional properties.