Probing the Electron Capture Dissociation Mass Spe

Authors: Doyong Kim PeiJing Pai Andrew J Creese Andrew W Jones David H Russell Helen J Cooper

Publish Date: 2015/04/02

Volume: 26, Issue: 6, Pages: 1004-1013

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Abstract

Electron capture dissociation mass spectrometry offers several advantages for the analysis of peptides most notably that backbone c and z fragments typically retain labile modifications such as phosphorylation We have shown previously that in some cases the presence of phosphorylation has a deleterious effect on peptide sequence coverage and hypothesized that intramolecular interactions involving the phosphate group were preventing separation of backbone fragments In the present work we seek to rationalize the observed ECD behavior through a combination of ECD of model peptides traveling wave ion mobility mass spectrometry and molecular dynamics simulations The results suggest that for doubly protonated ions of phosphopeptide APLpSFRGSLPKSYVK a saltbridge structure is favored whereas for the doublyprotonated ions of APLSFRGSLPKpSYVK ionic hydrogen bonds predominateElectron capture dissociation ECD is a tandem mass spectrometry technique in which trapped ions are irradiated with low energy electrons 1 2 ECD has proven to be highly useful in the analysis of peptides and proteins largely thanks to the retention of labile posttranslational modifications such as phosphorylation on the backbone c and z fragments 3 4 5 6 The approach has been applied to the largescale analysis of phosphoproteins 7In earlier work we demonstrated that the presence of phosphorylation can have a deleterious effect on the ECD fragmentation of doublyprotonated peptide ions 8 For peptides with sequence APLSFRGSLPKSYVK where each of the serines is variably phosphorylated ECD of the doubly charged precursor revealed no ECD fragments between the phosphoserine and either the lysine residue at position 11 or the arginine at position 6 We concluded that noncovalent interactions between the basic side chains and the phosphoserine were preventing separation of any ECD fragments that had formed The retention of noncovalent interactions following electron capture is well established 9 10 11 Support for this hypothesis is found in the work of Ruotolo et al 12 13 and Thalassinos et al 14 These researchers showed that collision cross sections CCS for some phosphopeptide ions differ from those for their unmodified counterparts Each of the studies reported negative deviations for the phosphopeptides ie CCS that fall below the random coil trend line This behavior suggests compaction of the conformation owing to intramolecular interactions between the phosphate group and protonated side chains of arginine or lysineHere we probe the nature of these noncovalent interactions either saltbridge or ionic hydrogen bond by use of a combination of traveling wave ion mobility spectrometry and molecular dynamics simulations Saltbridges are electrostatic interactions between amino acid residues of opposing charge and play important roles in protein stability 15 16 Iakoucheva et al have noted that basic residues lysine and arginine are found at high frequency in protein sequences near phosphorylation sites suggesting that saltbridges are important in phosphoprotein structures 17 It might be expected that any solutionphase saltbridge structures are destroyed as a result of proton transfer on transition to the gas phase 18 The question of whether solutionphase saltbridges are retained in the gas phase has been addressed by a number of researchers Williams and coworkers showed the most stable form of singly protonated peptide ions of bradykinin is the saltbridge structure 19 Further work by that group 20 revealed that for dipeptides of sequence XxxArg Xxx is Gly Val Pro Lys His or Arg the ArgArg peptide ion has a saltbridge structure whereas the remaining dipeptide ions have a single formal charge site suggesting that the presence of multiple basic residues stabilizes the saltbridge structure More recently one of our groups applied cryogenic ion mobilitymass spectrometry to demonstrate that the presence of solutionphase saltbridges aids retention of solutionphase structure in the gas phase 21ECD and the related technique of electron transfer dissociation have previously been applied to the investigation of intra and intermolecular electrostatic interactions in the gasphase structure of peptides and proteins Breuker et al applied ECD to the study of gasphase structure of the protein KIX 22 revealing that saltbridges and ionic hydrogen bonds conferred stability and to the gasphase unfolding of ubiquitin 23 again finding stabilization by saltbridges Vachet and coworkers showed via ETD experiments that many known solutionphase saltbridges were retained in the gasphase structures of protein ubiquitin CRABP1 azurin and β2microglobulin 24 Woods and coworkers have demonstrated the gasphase stability of the electrostatic interaction between arginine and phosphoserine in noncovalent complexes 25 and used ECD and ETD to identify the sites of interaction 26 The presence of phosphopeptide zwitterions has also been demonstrated by Hakansson and coworkers in their work on negative ion ECD 27The relationship between phosphopeptide structure and ECD behavior was probed by Tureček and coworkers 28 in their comparison of ECD ETD and ECID electron capture induced dissociation of doubly protonated phosphopeptides pSAAAR ApSAAR AApSAR and AAApSR They used a combination of molecular dynamics simulations and DFT calculations to obtain structures of the lowest energy conformers to explain the experimentally observed aberrant ECD fragmentation These phosphopeptides are simple enough to permit DFT calculations but complex enough that intramolecular interactions between side chains are anticipated In further work 29 they compared theoretical calculations of structure with results from drift tube ion mobility spectrometry and traveling wave ion mobility and IR action spectroscopy They found broad agreement between theory and experiment but could not directly assign structures to conformers because the differences in cross sections obtained for the three levels of theory were comparable to experimental error Unlike the peptides in this work Tureček’s peptides contain only one basic residue and the sites of protonation were the Arg sidechain and the Nterminus Deprotonation of the phosphate was considered in conjunction with protonation of an amide group however these structures collapsed with migration of the proton back to the phosphate

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