Sequence analysis of oligopeptides by secondary ion/collision activated dissociation mass spectrometry

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One Hundred Fifty-Seven nm photodissociation of singly protonated peptides generates unusual distributions of fragment ions. When the charge is localized at the C-terminus of the peptide, spectra are dominated by x-, v-, and w-type fragments. When it is sequestered at the N-terminus, a-and d-type ions are overwhelmingly abundant. Evidence is presented suggesting that the fragmentation occurs via photolytic radical cleavage of the peptide backbone at the bond between the ␣-and carbonyl-carbons followed by radical elimination to form the observed daughter ions. Low-energy fragmentation appears to be well described by the mobile proton model according to which vibrational excitation of the analyte leads to charge mobility [12]. Transfer of the charge proton to either the backbone carbonyl oxygen or amide nitrogen enables charge-induced cleavage of the peptide backbone. This process yields primarily b-and y-type fragment ions according to the standard nomenclature shown below [13,14].Higher energy activation methods involving collisions with gas molecules or surfaces can enable chargeremote fragmentation [15][16][17]. However, even in these cases, apparently, protonated peptides still generate some fragments through charge-directed processes [17]. Immobilization of the charge, either by using metal adducts [18] or charged chemical modifications [19 -22], has been used to inhibit charge-directed fragmentation. In these cases, primarily a-, d-, and w-type fragments are observed. Several mechanisms have been proposed for charge-remote peptide fragmentation, some involving homolytic radical cleavage [14,23].Electron capture dissociation (ECD) [24] and the recently reported electron-transfer dissociation (ETD)[25] appear to be nonthermal processes. These phenomena generate c-and z-type fragment ions upon the addition of an electron to a multiply charged protein or peptide ion. Two mechanisms have been proposed, one involving reactions of a free hydrogen atom generated when an electron is captured by a charged site on the analyte ion [26], and the other involving localization of the ϳ6 eV of energy that is generated upon charge neutralization. This energy then induces fragmentation through an excited electronic state [27]. Implicit in this second mechanism is the suggestion that techniques which excite appropriate electronic states can lead to unique, nonergodic fragmentation even for molecules as large as peptides and proteins [24,27]. This can occur if ions reach a dissociative electronic state and fragment before the energy is redistributed throughout the molecule. Electronic to vibrational relaxation, resulting in nonspecific excitation is an important competing process that often inhibits nonergodic processes, and rates of relaxation are predicted to increase with the size of the molecule [28].Lasers are powerful tools that can also be used to excite molecules to specific energy levels with either multiphoton or single photon processes. Light is not affected by the electric or magnetic fields of mass spectrometers, s...

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confidence: 99%

Pathways of Peptide Ion Fragmentation Induced by Vacuum Ultraviolet Light

Cui

Thompson

Reilly

2005

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“…The specific-fragmentation can be readily observed by MS n experiments to occur in an iterative fashion, suggesting that the C-terminal structure of the original [b (nϪ1) ϩ OH ϩ H] ϩ ion is maintained after subsequent rearrangement and fragmentation events in peptides which fragment specifically. A mechanism for the formation of specific-fragmenting and nonspecific-fragmenting andem mass spectrometry is a popular tool for the characterization of the primary structure of peptides© [1].©Much©effort©has©been©directed©to-ward the elucidation of the mechanisms of peptide fragmentation in the gas phase, and a number of reviews© on© the© subject© are© available© [2-© 4].© Some© details about© the mechanism© of© fragmentation© and© the structures of the fragmentation products have been elucidated© [5][6][7][8][9].©For©the©past©fifteen©years,©considerable effort has gone into the understanding of the structure peptide rearrangement ions and the mechanisms that drive© these© rearrangements© [10© -18].© One© of© the© major areas of© interest© has© been© the© [b (nϪ1) ϩ OH ϩ Cat] ϩ ion, which has been observed© in© a© variety© of peptides with a variety of cations using a variety of different ionization techniques© and© mass© analyzers© [12-14,© 16© -23].© Understanding© the structure of these rearrangement ions and their mechanisms of formation is important for the analysis of peptide tandem mass spectra,© as© well© as© for© helping© to© elucidate© the© general principles of peptide fragmentation.…”

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confidence: 99%

Formation of [b_(n−1)+OH+H]⁺ ion structural analogs by solution-phase chemistry

Sharp

Tomer

2005

Derivatization© of© a© variety© of© peptides© by© a method© known© to© enhance© anhydride© formation© is demonstrated by mass spectrometry to yield ions that have elemental composition and fragmentation properties identical to [b (nϪ1) ϩ ions formed by gas-phase rearrangement and fragmentation. The [b (nϪ1) ϩ ions formed by gas-phase rearrangement and fragmentation and the solution-phase [b (nϪ1) ϩ ion structural analogs formed by derivatization chemistry show two different forms of dissociation using multiple-collision CAD in a quadrupole ion trap and unimolecular decomposition in a TOF-TOF; one group yields identical product ions as a truncated form of the peptide with a free C-terminal carboxylic acid and fragments at the same activation energy; the other group fragments differently from the truncated peptide, being more resistant to fragmentation than the truncated peptide and yielding primarily the [b (nϪ2) ϩ OH ϩ H] ϩ product ion. Nonergodic electron capture dissociation MS/MS suggests that any structural differences between the specific-fragmenting [b (nϪ1) ϩ OH ϩ H] ϩ ions and the truncated peptide is at the C-terminus of the peptide. The specific-fragmentation can be readily observed by MS n experiments to occur in an iterative fashion, suggesting that the C-terminal structure of the originalϩ ion is maintained after subsequent rearrangement and fragmentation events in peptides which fragment specifically. A mechanism for the formation of specific-fragmenting and nonspecific-fragmenting [b (nϪ1) ϩ ion©was hypothesized to be identical to that of the©truncated peptide,©i.e.,©a©free©acid©C-terminus© [12].©Multiple groups© have proposed a mechanism for the formation of the [b (nϪ1) ϩ OH ϩ H] ϩ that involves the initial formation of a seven-membered hydrogen-bonded ring at the neutral C-terminus, followed by collapse into a five-membered cyclic anhydride, which then results in loss© of© the© C-terminal© amino© acid© residue© with© retention of© the original C-terminal© oxygen© at© the new© C-terminal free© acid© (Scheme© 1)© [12,© 13].© Two© important© features© of this mechanism should be noted-the initial formation of a five-membered cyclic anhydride and the free carboxylic acid C-terminus of the end product.Recently, Farrugia and O'Hair reported the results of experimentation and computational modeling of

“…The pseudo-MS 3 technique used in this study has some limitations with respect to sample purity, because there is no step of mass selection before the first stage of collisional activation; however, it has the advantage that a standard triple quadrupole instrumentation can be used. (J Am Soc Mass Spectrom 1998, 9, 606 -611) © 1998 American Society for Mass Spectrometry U nder the conditions of collision-induced dissociation (CID) peptide molecular ions dissociate at different sites of the peptide backbone [1][2][3]. This effect results in various types of fragment ions [4].…”

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confidence: 99%

Single series peptide fragment ion spectra generated by two-stage collision-induced dissociation in a triple quadrupole

Lehmann

1998

By using nanoelectrospray ionization and a triple quadrupole analyzer, simplified fragment ion spectra of peptides have been recorded by combining skimmer collision-induced dissociation with precursor ion scanning or neutral loss scanning. These pseudo-MS 3 scan modes are characterized by two-stage collision-induced dissociation and have been termed sCID/ precursor and sCID/neutral loss scan, respectively. By these scan modes, peptide fragment ion spectra can be generated that predominantly show signals of a single fragment ion series, such as the B or YЉ series. Skimmer collision-induced dissociation combined with scanning for neutral loss of 28 generates spectra showing B ions, whereas combination with precursor ion scanning for the YЉ 1 ion results in spectra showing YЉ ions for tryptic peptides (YЉ 1 ϭ m/z 147 for C-terminal lysine, YЉ 1 ϭ m/z 175 for C-terminal arginine). Sequence information including the direction of the sequence is easily extracted from the simplified fragment ion spectra generated by two-stage collision-induced dissociation, because the scan mode defines the type of fragments observed. The analytical results reported are similar to those that have been achieved in MS 3 experiments using a hybrid BEQQ or a pentaquadrupole mass spectrometer (Schey, K. L.; Schwartz, J. C.; Cooks, R. G. Rapid Commun. Mass Spectrom. 1989, 3, 305-309). The pseudo-MS 3 technique used in this study has some limitations with respect to sample purity, because there is no step of mass selection before the first stage of collisional activation; however, it has the advantage that a standard triple quadrupole instrumentation can be used. (J Am Soc Mass Spectrom 1998, 9, 606 -611) © 1998 American Society for Mass Spectrometry U nder the conditions of collision-induced dissociation (CID) peptide molecular ions dissociate at different sites of the peptide backbone [1][2][3]. This effect results in various types of fragment ions [4]. Sequence information is provided by the mass differences between adjacent ions of a single fragment ion series. However, a peptide product ion spectrum generally consists of several fragment ion series superimposed on each other, a situation that seriously hampers the extraction of sequence information. Several strategies have been introduced for the identification of fragment ion series, in particular for discrimination between N-terminal and C-terminal fragment ions. For this purpose, e.g., selective derivatization of the Nterminal amino function by a quaternary reagent [5], analyzing the stability of fragment ions [6], esterification of the C-terminal carboxyl function, selective labeling of the C-terminus by protease-catalyzed incorporation of oxygen-18 [7-9], or MS 3 experiments involving reaction intermediate scanning [10] have been used.In the past, MS/MS analyses involving more than one step of collisional activation have been performed with hybrid instruments [10,11], pentaquadrupole instruments [10,12], quadrupole ion trap analyzers [13,14], or magnetic ion traps [15,16]. In the fo...