Effects of Charge State and Cationizing Agent on the Electron Capture Dissociation of a Peptide

Electron capture dissociation at 86 K of the linear peptide Substance P produced just two backbone fragments, whereas at room temperature eight backbone fragments were formed. Similarly, with the cyclic peptide gramicidin S, just one backbone fragment was formed at 86 K but five at room temperature. The observation that some backbone scissions are active and others inactive, when all involve N™C ␣ cleavages and have a high rate constant, indicates that the more specific fragments at low temperatures reflects the reduced conformation heterogeneity at low temperatures. This is supported by reduced or inactive hydrogen loss, a channel that has previously been shown to be affected by conformation. The conclusion that the ECD fragments are a snapshot of the conformational (intramolecular solvation shell) heterogeneity helps explain how the relative intensities of ECD fragments can be different on different instrument and highlights the common theme in methodologies used to increase sequence coverage, namely an increase in the conformational heterogeneity of the precursor ion population. ( ploys reactions of multiprotonated peptides/ proteins with low energy electrons to generate peptide fragments. The high sequence coverage and the ability to retain labile groups after ECD have allowed posttranslational modifications (PTMs) and point mutations (PMs) in peptides and proteins to be both identified and localized. Such performance is of great value for the analysis of the proteome, particularly that of diseased organisms. PTMs and PMs are widespread and are frequently associated with disease [5][6][7], for example over 80 different point mutations have been found in transthyretin (most of which lead to autosomal disorders) [8].To be able to fully characterize such modified proteins, 100% sequence coverage is required (so-called top-down proteomics). Several methods have been developed to increase the sequence coverage of ECD, which can be grouped into either infrared illumination [9 -11] or collisional activation [12]. These methods increase the average internal energy of the ions, which also affects the conformation of these gas-phase ions [13].There is experimental and theoretical evidence that ECD is directed by the internal solvation of the (neutralized) proton [9, 14 -18]. If the reaction progresses faster than the electron-proton recombination energy is randomized and thermal fluctuations are small, specific fragments would be expected from a single (frozen) conformer, reflecting the solvation shell, specific for that conformation, surrounding the neutralized proton. Thermal fluctuations that result in a dynamic solvation shell, but not conformational change (local fluctuations rather than a global change), would produce a greater number of fragments. Finally, multiple conformations as well as thermal fluctuations would produce yet more fragments. Because the methods used to increase the sequence coverage of ECD all increase the internal energy of the ions, and so affect the conformation of the gas-phase ions, the...

“…. not due to effects of tertiary structure" [29]. This behavior is in stark contrast to the cold temperature results reported here and several previous studies.…”

Section: Resultscontrasting

confidence: 92%

Electron capture dissociation at low temperatures reveals selective dissociations

Mihalca

Kleinnijenhuis

McDonnell

et al. 2004

“…The RE is calculated from the ionization potential of the atom, its cation affinity, and its neutral atom affinity towards singly charge-reduced peptides. Although it is difficult to precisely calculate the affinities, Williams and coworker [24] provided the estimated recombination energy of the charge carrying hydrogen, lithium, and cesium atoms. The RE for sodium is likely between 72 and 85 kcal/mol, the values corresponding to cesium and lithium, respectively.…”

Section: Discussionmentioning

confidence: 99%

Sulfopeptide fragmentation in electron-capture and electron-transfer dissociation

Medzihradszky

Guan

Maltby

et al. 2007

Sulfopeptides can be misassigned as phosphopeptides because of the isobaric nature of the sulfo-and the phosphomoieties. Instruments having the ability to measure mass with high accuracy may be employed to distinguish these moieties based on their mass defect (the sulfo-group is 9 mmu lighter than the phosphomoiety). However, the assignment of the exact site(s) of post-translational modification is required to probe biological function. We have reported earlier that peptides with identical sequences containing either O-sulfo-or Ophospho-modifications display different fragmentation behavior (K. F. Medzihradszky et al., Mol. Cell. Proteom. 2004, 3, 429 -440). We have also established that O-sulfo moieties are susceptible to side-chain fragmentation during collision-induced dissociation. Our present study provides evidence that neutral SO 3 losses can also occur in electron capture dissociation and electron-transfer dissociation experiments. We also report that such neutral losses may be reduced by fragmenting peptide-alkali metal adducts, such as sodiated or potassiated peptides. ( -5]. Sulfopeptides also display an 80 Da mass increase as well as similar chromatographic and mass spectrometric behavior [6]. The sulfopeptides are 9 mmu lighter than their phosphorylated counterparts. They can easily be misidentified as phosphopeptides. However, the most significant difference between phosphoand sulfopeptides is revealed during MS/MS analysis. In CID analyses phosphorylated Tyr residues retain the modification, and even their modified immonium ion is sometimes observed, while phosphorylated Ser and Thr residues undergo ␤-elimination of H 3 PO 4 . Thus, the fragments detected in the CID spectra of phosphopeptides either display a ϩ80 Da or a Ϫ18 Da mass shift in comparison to the fragments of an unmodified peptide. Under identical conditions, collisional activation of Osulfopeptides leads to a gas-phase rearrangement reaction that completely eliminates the sulfate from the molecular ion, and the CID fragment spectrum is practically identical to that observed for the unmodified molecule [6]. We have analyzed numerous O-sulfopeptides and have not detected any CID fragments that still contained the sulfate group.While the different CID behavior proved to be a reliable tool for the differentiation of these two isobaric modifications, the gas-phase elimination of the sulfomoiety also prevented the assignment of the modification site. Therefore, it seemed essential to evaluate electron-capture dissociation based on reports that most of the labile side chains of peptides and proteins studied remain intact [7]. Electron-transfer dissociation [8,9] was also employed to analyze synthetic phospho-and sulfopeptides. Here we report our observations on the ECD and ETD fragmentation of sulfopeptides. Experimental MaterialsPhosphopeptides and sulfopeptides were synthesized as described earlier [6]. Their sequences are as follows: RIEVALsTK; RIEVALpTK; LAGLQDEIGsSLR; LAGLQDEIGpSLR; Ac-LAGLQDEIGsSLR-amide. Caerulein, ϽQQDsYTGWMDF-ami...

“…For multiply protonated proteins, this recombination energy resulting from EC has been estimated to be 4 -7 eV [7,15]. The recombination energy for protonated, lithiated, and cesiated glycine decreases with increasing cation size [38]. The fragment ions formed by ECD of peptides that are cationized with two different cations are consistent with the preferred neutralization of the cation of highest recombination energy [38].…”

mentioning

confidence: 89%

Nonergodicity in electron capture dissociation investigated using hydrated ion nanocalorimetry

Leib

Donald

Bush

et al. 2007

Self Cite

118

Hydrated divalent magnesium and calcium clusters are used as nanocalorimeters to measure the internal energy deposited into size-selected clusters upon capture of a thermally generated electron. The infrared radiation emitted from the cell and vacuum chamber surfaces as well as from the heated cathode results in some activation of these clusters, but this activation is minimal. No measurable excitation due to inelastic collisions occurs with the low-energy electrons used under these conditions. Two different dissociation pathways are observed for the divalent clusters that capture an electron: loss of water molecules (Pathway I) and loss of an H atom and water molecules (Pathway II). For Ca(H 2 O) n 2ϩ , Pathway I occurs exclusively for n Ն 30 whereas Pathway II occurs exclusively for n Յ 22 with a sharp transition in the branching ratio for these two processes that occurs for n Ϸ 24. The number of water molecules lost by both pathways increases with increasing cluster size reaching a broad maximum between n ϭ 23 and 32, and then decreases for larger clusters. From the number of water molecules that are lost from the reduced cluster, the average and maximum possible internal energy is determined to be ϳ4.4 and 5.2 eV, respectively, for Ca(. This value is approximately the same as the calculated ionization energies of M(H 2 O) n ϩ , M ϭ Mg and Ca, for large n indicating that the vast majority of the recombination energy is partitioned into internal modes of the ion and that the dissociation of these ions is statistical. For smaller clusters, estimates of the dissociation energies for the loss of H and of water molecules are obtained from theory. For Mg(H 2 O) n 2ϩ , n ϭ 4 -6, the average internal energy deposition is estimated to be 4.2-4.6 eV. The maximum possible energy deposited into the n ϭ 5 cluster is Ͻ7.1 eV, which is significantly less than the calculated recombination energy for this cluster. There does not appear to be a significant trend in the internal energy deposition with cluster size whereas the recombination energy is calculated to increase significantly for clusters with fewer than 10 water molecules. These, and other results, indicate that the dissociation of these smaller clusters is nonergodic. . The effectiveness of the bottom-up method for complex samples can be enhanced by using multidimensional separations. Clemmer and coworkers elegantly demonstrated that combining on-line liquid chromatography (LC) with ion mobility spectrometry and MS can greatly improve separations without increasing analysis times over LC/MS alone [2][3][4]. In contrast, the "top-down" approach to protein characterization has the advantage that de novo sequencing, including the identification and structural localization of labile posttranslational modifications, can be done directly on protein mixtures without proteolysis [5,6]. This top-down approach has greatly benefited from the development of electron capture dissociation (ECD), a method pioneered by McLafferty and coworkers [6][7][8][9]. In a typical ECD experim...