Investigation into reversed-phase chromatography peptide separation systems part IV : characterisation of mobile phase selectivity differences

The differentiation of mobile phase compositions between sub-classes which exhibit distinct chromatographic selectivity (i.e. termed characterisation) towards a range of peptide probes with diverse functionality and hence the possibility for multi-modal retention mechanisms has been undertaken. Due to the complexity of peptide retention mechanisms in given mobile phase conditions, no attempt has been made to explain these, instead mobile phases have simply been classified into distinct groups with an aim of identifying those yielding differing selectivities for use in strategic method development roadmaps for the analysis of peptide mixtures. The selectivity differences between nine synthetic peptides (fragments of [Ile27]-Bovine GLP-2) were used to assess how fifty-one RPC mobile phase compositions of differing pH (range 1.8 – 7.8), salt types, ionic strengths, ion-pair reagents and chaotropic / kosmotropic additives affected chromatographic selectivity on a new generation C18 stationary phase (Ascentis Express C18). The mobile phase compositions consisted of commonly used and novel UV or MS compatible additives. The chemometric tool of Principal Component Analysis (PCA) was used to visualise the differences in selectivity generated between the various mobile phases evaluated. The results highlight the importance of screening numerous mobile phases of differing pH, ion-pair reagents and ionic strength in order to maximise the probability of achieving separation of all the peptides of interest within a complex mixture. PCA permitted a ranking of the relative importance of the various mobile phase parameters evaluated. The concept of using this approach was proven in the analysis of a sample of Bovine GLP-2 (1-15) containing synthesis related impurities. Mobile phases with high ionic strength were demonstrated to be crucial for the generation of symmetrical peaks. The observations made on the C18 phase were compared on three additional stationary phases (i.e. alkyl amide, fluorophenyl and biphenyl), which had previously been shown to possess large selectivity differences towards these peptides, on a limited sub-set of mobile phases. With the exception of the ion-pair reagent, similar trends were obtained for the C18, fluorophenyl and biphenyl phases intimating the applicability of these findings to the vast majority of RPC columns (i.e. neutral or weakly polar in character) which are suitable for the analysis of peptides. The conclusions were not relevant for columns with a more disparate nature (i.e. containing a high degree of positive charge)

Tandem MALDI/EI ionization for tandem Fourier transform ion cyclotron resonance mass spectrometry of polypeptides

Abstract The tandem ionization (TI) technique in which protonated polypeptides [M + H] + produced by matrix-assisted laser desorption ionization (MALDI) are further ionized inside a Fourier transform (FT) mass spectrometer by >10 eV electrons has been extended to allow the formed radical dications [M + H] •2+ to capture low energy electrons. The capture produces [M + H] + * ions which, being isoelectronic with the precursor ions, are electronically excited. The electron capture process is >10 eV exothermic and may cause fragmentation, mainly into even-electron a and c ions. This fragmentation bears similarity with UV photodissociation, but produces little secondary fragmentation. Up to date, 10 peptides from 1 to 3 kDa have been subjected to this electronic excitation dissociation (EED). The details and potential analytical applications of EED are discussed

Probing the Binding Interfaces of Protein Complexes Using Gas-Phase H/D Exchange Mass Spectrometry

SummaryFast gas-phase hydrogen/deuterium exchange mediated by ND3 gas and measured by mass spectrometry (gas-phase HDX-MS) is a largely unharnessed, fast, and sensitive method for probing primary- and higher-order polypeptide structure. Labeling of heteroatom-bound non-amide hydrogens in a sub-millisecond time span after electrospray ionization by ND3 gas can provide structural insights into protein conformers present in solution. Here, we have explored the use of gas-phase HDX-MS for probing the higher-order structure and binding interfaces of protein complexes originating from native solution conditions. Lysozyme ions bound by an oligosaccharide incorporated less deuterium than the unbound ion. Similarly, trypsin ions showed reduced deuterium uptake when bound by the peptide ligand vasopressin. Our results are in good agreement with crystal structures of the native protein complexes, and illustrate that gas-phase HDX-MS can provide a sensitive and simple approach to measure the number of heteroatom-bound non-amide side-chain hydrogens involved in the binding interface of biologically relevant protein complexes