Fragmentation reactions of protonated peptides containing glutamine or glutamic acid

Some of the most prominent "neutral losses" in peptide ion fragmentation are the loss of ammonia and water from N-terminal glutamine. These processes are studied by electrospray ionization mass spectrometry in singly-and doubly-protonated peptide ions undergoing collision-induced dissociation in a triple quadrupole and in an ion trap instrument. For this study, four sets of peptides were synthesized: (1) QLLLPLLLK and similar peptides with K replaced by R, H, or L, and Q replaced by a number of amino acids, (2) QL n K (n ϭ 0, 1, 3, 5, 7, 9, 11), (3) QL n R (n ϭ 0, 1, 3, 5, 7, 9), and (4) QL n (n ϭ 1, 2, 3, 4, 8). The results for QLLLPLLLK and QLLLPLLLR show that the singly protonated ions undergo loss of ammonia and to a smaller extent loss of water, whereas the doubly protonated ions undergo predominant loss of water. The fast fragmentation next to P (forming the y 5 ion) occurs to a larger extent than the neutral losses from the singly protonated ions but much less than the water loss from the doubly protonated ions. The results from these and other peptides show that, in general, when N-terminal glutamine peptides have no "mobile protons", that is, the number of charges on the peptide is no greater than the number of basic amino acids (K, R, H), deamination is the predominant neutral loss fragmentation, but when mobile protons are present the predominant process is the loss of water. Both of these processes are faster than backbone fragmentation at the proline. These results are rationalized on the basis of resonance stabilization of the two types of five-membered ring products that would be formed in the neutral loss processes; the singly protonated ion yields the more stable neutral pyrrolidinone ring whereas the doubly protonated ion yields the protonated aminopyrroline ring (see Schemes). The generality of these trends is confirmed by analyzing an MS/MS spectra library of peptides derived from tryptic digests of yeast. In the absence of mobile protons, glutamine deamination is the most rapid neutral loss process. For peptides with mobile protons, dehydration from glutamine is far more rapid than from any other amino acid. Most strikingly, end terminal glutamine is by far the most labile source of neutral loss in excess-proton peptides, but not highly exceptional when mobile protons are not available. In addition, rates of deamination are faster in lysine versus arginine C-terminus peptides and 20 times faster in positively charged than negatively charged peptides, demonstrating that these formal neutral loss reactions are not "neutral reactions" but depend on charge state and stability. (J Am Soc Mass Spectrom 2007, 18, 27-36)

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confidence: 67%

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confidence: 99%

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confidence: 99%

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Dehydration versus deamination of N-terminal glutamine in collision-induced dissociation of protonated peptides

Neta

Kilpatrick

et al. 2007

“…Whereas most N-terminal residues lead to similar behavior, E and Q lead to much smaller intensities for the y n-2 ion peaks, but the difference decreases with increased peptide length (as reported before for a specific example [4]). Since N-terminal E and Q can undergo loss of water or ammonia to form pyroglutamic acid [9,10], we examined the importance of these losses. It has been reported that tryptic peptides with N-terminal glutamine undergo loss of either water or ammonia when singly protonated, but predominantly loss of water when doubly protonated [10].…”

Section: Resultsmentioning

confidence: 99%

Effect of N-terminal glutamic acid and glutamine on fragmentation of peptide ions

Godugu¹,

Neta²,

Simón‐Manso³

et al. 2010

A prominent dissociation path for electrospray generated tryptic peptide ions is the dissociation of the peptide bond linking the second and third residues from the amino-terminus. The formation of the resulting b 2 and y n-2 fragments has been rationalized by specific facile mechanisms. An examination of spectral libraries shows that this path predominates in diprotonated peptides composed of 12 or fewer residues, with the notable exception of peptides containing glutamine or glutamic acid at the N-terminus. To elucidate the mechanism by which these amino acids affect peptide fragmentation, we synthesized peptides of varying size and composition and examined their MS/MS spectra as a function of collision voltage in a triple quadrupole mass spectrometer. Loss of water from N-terminal glutamic acid and glutamine is observed at a lower voltage than any other fragmentation, leading to cyclization of the terminal residue. This cyclization results in the conversion of the terminal amine group to an imide, which has a lower proton affinity. As a result, the second proton is not localized at the N-terminus but is readily transferred to other sites, leading to fragmentation near the center of the peptide. eptide ion fragmentation by tandem mass spectrometry has become a routine means of determining peptide sequence for the purpose of protein identification [1]. A prominent dissociation path for electrospray-generated tryptic peptide ions is the breaking of the peptide bond linking the second and third residues from the amino-terminus [2-6]. The formation of the resulting b 2 and y n-2 fragments from diprotonated peptide ions (with n amino acid residues) has been rationalized by specific facile mechanisms [2]. It has been pointed out, however, that certain peptides ions do not fragment preferentially by this route but rather fragment at peptide bonds closer to the center of the peptide [2]. To explain the difference between the two groups of peptides it was suggested that the dominant b 2 ions formed from the first group have a protonated diketopiperazine structure (1) whereas b 2 ions from the other peptides have a protonated oxazolone structure (2).More recent studies, however, indicated that b 2 ions from the first group also have the oxazolone structure [5,6] so that the reason for the difference between the two groups remains unclear. Another recent study of several synthetic (Ala) x His peptide ions indicated that this fragmentation pathway diminished with increasing peptide length [4].In the present study, we attempt to distinguish between these two groups of peptides by searching for correlation between the mode of fragmentation and the amino acid sequence, mainly the amino acids at or near the N-terminus. After finding certain differences through statistical analysis of a large database of peptide MS/MS spectra, we synthesized specific peptides to study their fragmentation as a function of collision Address reprint requests to Dr.

“…For peptides with amino acids such as Lys (K) and Arg (R), the basic side chain can influence peptide dissociation by participating in nucleophilic attacks [20 -23], providing stabilization through salt bridges [23,24], or by facilitating proton transfers. Peptides with Asn (N) and Gln (Q) residues, which feature amide side chains, can experience attack by the side-chain amide oxygen on the carbon center of the amide bond to form a cyclic isoimide, which is a nonclassic b ion [20,[25][26][27]. Alternatively, another nonclassic b ion can arise via attack of the side-chain amide nitrogen on the carbon center of the amide bond leading to a six-membered ring structure (glutarimide) such as described by Jonsson et al [25] and Farrugia and coworkers [20].…”

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confidence: 99%

Influence of amino acid side chains on apparent selective opening of cyclic b₅ ions

Molesworth

Osburn

Stipdonk

2010

In this study, the possible influence of acidic, basic, and amide side chains on the opening of a putative macrocyclic b ion (b 5 ϩ ) intermediate was investigated. Collision induced dissociation (CID) of b 5 ions was studied using a group of hexapeptides in which amino acids with the side chains of interest occupied internal sequence positions. Further experiments were performed with permuted isomers of glutamine (Q) containing peptides to probe for sequence scrambling and whether the specific sequence site of the residues influences opening of the macrocycle. Overall, the trend for (apparent) preferential/selective opening of the cyclic b 5 ϩ , presumably due to the side chain, followed by the loss of the amino acid with active side group is: [3], is dependent, in part, on product ion distributions generated by CID. For bioinformatics approaches in particular, prediction of fragmentation patterns often employs rules that are rudimentary and simple. This may lead to invalid assignments of peptide and protein identity [3,4]. Certainly, a better understanding of fundamental gas-phase peptide fragmentation chemistry and physics would potentially lead to enhanced and more accurate bioinformatics-based MS/MS sequencing.Using low-energy collision induced dissociation (CID), fragmentation of protonated peptides traditionally involves charge (proton) mediated reactions, with induced cleavage of amide bonds leading to the generation of b, y, and a ions [5,6]. Development of the mobile proton model [7,8] of peptide fragmentation, and related amide bond cleavage pathways [9 -14], has been focused on the energetics and kinetics of proton mobilization. The more recent pathways in competition (PIC) fragmentation model [14] uses the mobile proton model as a foundation for understanding, but takes into account the structures and reactivity of key reactive configurations and primary fragments as well as transition states and their energies.There is a great deal of evidence that N-terminal b n type fragment ions have structures that include, at least in part, C-terminal oxazolone rings [9,15], and retain much of the primary sequence of the precursor peptide ion. However, more recent experiments [16 -19] strongly suggest that a macro-cyclic b ion isomer, or intermediate, can arise through cyclization of the linear, oxazolone-terminated b ions; this macrocyclic species can then open at different amide bonds to regain a linear, oxazolone terminated structure. One problematic outcome of such a cyclization and reopening process is the associated scrambling of the original primary sequence. This type of pathway is referred to as b-type scrambling of peptide fragment ions [16]. Above and beyond the "head to tail" type formation of the macrocycle, there exist several other possible processes that could play a significant role in the scrambling of sequence, the majority of which involve opening of the cyclic b ion. For example, peptides that contain acidic, basic, and amide side amino acids feature side chains that can serve as nucleo...