Gas-phase fragmentation of protonated mono-N-methylated peptides. Analogy with solution-phase acid-catalyzed hydrolysis

A series of calculations, varying from simple electrostatic to more detailed semi-empirical based molecular dynamics ones, were carried out on charged gas phase ions of the cytochrome c dimer. The energetics of differing charge states, charge partitionings, and charge configurations were examined in both the low and high charge regimes. As well, preliminary free energy calculations of dissociation barriers are presented. It is shown that one must always consider distributions of charge configurations, once protein relaxation effects are taken into account, and that no single configuration dominates. All these results also indicate that in the high charge limit, the dissociation of protein complex ions is governed by electrostatic repulsion from the net charges, the consequences of which are enumerated and discussed. There are two main trends deriving from this, namely that charges will move so as to approximately maintain constant surface charge density, and that the lowest barrier to dissociation is the one that produces fragment ions with equal charges. In particular, it is shown that the charge-to-mass ratio of a fragment ion is not the key physical parameter in predicting dissociation products. In fact, from the perspective of the division of total charge, many dissociation pathways reported to be "asymmetric" in the literature should be more properly labelled as "symmetric" or "near-symmetric". The Coulomb repulsion model assumes that the timescale for charge transfer is faster than that for protein structural changes, which in turn is faster than that for complex dissociation. One challenge in using mass spectrometry for the general analysis of protein complexes is understanding the dissociation mechanism of these complexes in the gas phase. Many groups [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] have reported asymmetric dissociation behaviour for large multimeric proteins. A small subunit, typically a protein monomer, is ejected from the complex during dissociation, with the monomer carrying away a disproportionate amount of charge for its relative mass. Understanding the cause of this phenomenon will help to improve our understanding of the structural properties of noncovalent complexes.It is important to investigate how these dissociations occur and the factors that influence them. Using Fourier-transform mass spectrometry (FTMS), Jurchen and Williams [6] have conducted a number of studies in order to understand the origin of these charge distributions. In these experiments, isolated charge states of cytochrome c dimer were dissociated by sustained off-resonance irradiation collisionally activated dissociation (SORI-CAD). According to Jurchen and Williams [6], the asymmetric charge distribution depends upon charge state, dissociation energy, and conformational flexibility. These studies showed that higher charge states lead to symmetric charge products while lower charge states lead to an asymmetric charge dissociation pathway. Further, their results with different excitation energies show ...

Section: Application Of the Coulomb Repulsion Modelmentioning

confidence: 99%

Theoretical investigations of the dissociation of charged protein complexes in the gas phase

Wanasundara

Thachuk

2007

“…The typical b n ion structure produced when a charge induces cleavage is an oxazalone. This protonated oxazalone forms when a backbone carbonyl oxygen attacks°an°electropositive°backbone°carbonyl°carbon° [47][48][49][50][51][52]°(Scheme°2).°It°is°not°clear°what°structure°the°b n ions have in the absence of an added proton. The lack of *a n ions°observed°in°Figure°4a°indicates°the°*b n ions observed by selective cleavage do not have the same *b n structure as formed from P ϩ LDIFSDF(OMe) 3 and suggests the ions are forming in a mechanistically different manner.…”

Section: Fragmentation Of P ϩ Ldifsdf and P ϩ Ldifsdf(ome)mentioning

confidence: 99%

“…The Asp acidic hydrogen can be considered mobile "locally" in this mechanism because it is transferred to the backbone carbonyl or amide nitrogen during formation of the anhydride b-ion. This anhydride b-ion differs from the "typical" b-ion structure formed when a backbone carbonyl oxygen attacks an electropositive backbone carbonyl carbon to form a protonated oxazalone structure [47][48][49][50][51][52], as shown Scheme 2.…”

mentioning

confidence: 98%

Computational investigation and hydrogen/deuterium exchange of the fixed charge derivative tris(2,4,6-Trimethoxyphenyl) phosphonium: Implications for the aspartic acid cleavage mechanism

Herrmann

Wysocki

Vorpagel

2005

Aspartic acid (Asp)-containing peptides with the fixed charge derivative tris(2,4,6-trimethoxyphenyl) phosphonium (tTMP-P ϩ ) were explored computationally and experimentally by hydrogen/deuterium (H/D) exchange and by fragmentation studies to probe the phenomenon of selective cleavage C-terminal to Asp in the absence of a "mobile" proton. Ab initio modeling of the tTMP-P ϩ electrostatic potential shows that the positive charge is distributed on the phosphonium group and therefore is not initiating or directing fragmentation as would a "mobile" proton. Geometry optimizations and vibrational analyses of different Asp conformations show that the Asp structure with a hydrogen bond between the side-chain hydroxy and backbone carbonyl lies 2.8 kcal/mol above the lowest energy conformer. In reactions with D 2 O, the phosphonium-derived doubly charged peptide (H ϩ )P ϩ LDIFSDF rapidly exchanges all 12 of its exchangeable hydrogens for deuterium and also displays a nonexchanging population. With no added proton, P ϩ LDIFSDF exchanges a maximum of 4 of 11 exchangeable hydrogens for deuterium. No exchange is observed when all acidic groups are converted to the corresponding methyl esters. Together, these H/D exchange results indicate that the acidic hydrogens are "mobile locally" because they are able to participate in exchange even in the absence of an added proton. Fragmentation of two distinct (H ϩ )P ϩ LDIFSDF ion populations shows that the nonexchanging population displays selective cleavage, whereas the exchanging population fragments more evenly across the peptide backbone. This result indicates that H/D exchange can sometimes distinguish between and provide a means of separation of different protonation motifs and that these protonation motifs can have an effect on the fragmentation. (J Am Soc Mass Spectrom 2005, 16, 1067-1080

“…Amide nitrogen methylation occurs in nature, such as in bacterial and fungal peptides/proteins that have possible medicinal properties [14 -17], and has been used as a tool for protein binding studies [18]. The dissociation behavior of N-methylated peptide ions subjected to collisional activation has been reported, and it was noted that there was an increase in relative abundance of the products coming from cleavage of the amide bond at the methylation site [19,20]. It was suggested that this preferred cleavage occurred due to the increased basicity of the amide nitrogen.…”

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confidence: 99%

Electron transfer dissociation of amide nitrogen methylated polypeptide cations

Crizer

McLuckey

2009

Unmodified and amide nitrogen methylated peptide cations were reacted with azobenzene radical anions to study the utility of electron transfer dissociation (ETD) in analyzing N-methylated peptides. We show that methylation of the amide nitrogen has no deleterious effects on the ETD process. As a result, location of alkylation on amide nitrogens should be straightforward. Such a modification might be expected to affect the ETD process if hydrogen bonding involving the amide hydrogen is important for the ETD mechanism. The partitioning of the ion/ion reaction products into all of the various reaction channels was determined and compared for modified and unmodified peptide cations. While subtle differences in the relative abundances of the various ETD channels were observed, there is no strong evidence that hydrogen bonding involving the amide nitrogen plays an important role in the ETD process. (J Am Soc