Probing Proline Effect in CID of Protonated Peptides

Vaisar, Tomáš; Urban, Ján

doi:10.1002/(sici)1096-9888(199610)31:10<1185::aid-jms396>3.0.co;2-q

Cited by 170 publications

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“…Preferential cleavage N-terminal to Pro in tryptic peptides has been noted previously [9,16]. Vaisar and Urban [27] have reported that singly-protonated pentapeptides with Pro in the central position show enhanced cleavage N-terminal to Pro (to produce y 3 ). They also reported that replacement of Pro with Sar in these peptides also resulted in enhanced cleavage to produce y 3 although not to the extent that Pro did.…”

Section: Resultsmentioning

confidence: 68%

Effect of the Identity of Xaa on the Fragmentation Modes of Doubly-Protonated Ala-Ala-Xaa-Ala-Ala-Ala-Arg

Harrison

2011

J. Am. Soc. Mass Spectrom.

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The product ion mass spectra resulting from collisional activation of doubly-protonated tryptic-type peptides Ala-Ala-Xaa-Ala-Ala-Ala-Arg have been determined for Xaa = Ala(A), Ser(S), Val(V), Thr(T), Ile(I), Phe(F), Tyr(Y), Sar, Met(M), Trp(W), Pro(P), and Gln(Q). The major fragmentation reaction involves cleavage of the second amide bond (counting from the N-terminus) except for Xaa = Ser and Thr where elimination of H 2 O from the [M + 2H] +2 ion forms the base peak. In general, the extent of cleavage of the second amide bond shows little dependence on the identity of Xaa and little dependence on whether the bond cleavage involves symmetrical bond cleavage to form a y 5 /b 2 ion pair or asymmetrically to form y 5 +2 and a neutral b 2 species. Notable exceptions to this generalization occur for Xaa equal to Pro or Sar. For Xaa = Pro only cleavage of the second amide bond is observed, consistent with a pronounced proline effect, i.e., cleavage N-terminal to Pro. When Xaa = Sar considerably enhanced cleavage of the second amide bond also is observed, suggesting that at least part of the proline effect relates to the tertiary nature of the amide nitrogen. In the competition between symmetric and asymmetric bond cleavage an attempt to establish a linear free energy correlation in relating ln(y 5 +2 /y 5 ) to PA(H-Xaa-OH) did not lead to a reasonable correlation although the trend of increasing y 5 +2 /y 5 ratio with increasing proton affinity of H-Xaa-OH was clear. Proline showed a unique behavior in giving a much higher y 5 +2 /y 5 ratio than any of the other residues studied.

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Section: Resultsmentioning

confidence: 68%

Effect of the Identity of Xaa on the Fragmentation Modes of Doubly-Protonated Ala-Ala-Xaa-Ala-Ala-Ala-Arg

Harrison

2011

J. Am. Soc. Mass Spectrom.

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“…Cleavage of the amide bond N-terminal to a proline residue is a preferred pathway in CID fragmentation of peptides (the proline effect). [20,22] Additionally, loss of the two N-terminal residues from a peptide is one of the very common fragmentation pathways in CID. [23] Given these theories and observations, CID fragmentation of doubly protonated IgG1 T27 to form a single dominant y 15 2+ (PPVLDSDGSFFLYSK) product ion is not difficult to interpret.…”

Section: Specificity For Confirmation Of Etanerceptmentioning

confidence: 99%

Confirmatory analysis of etanercept in equine plasma by LC‐MS for doping control

Guan

Robinson

Soma

2016

Drug Testing and Analysis

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Etanercept is a protein-based medication for the treatment of human patients with rheumatoid arthritis and other autoimmunebased diseases; its pharmacological action is to inhibit and antagonize tumour necrosis factor alpha. Etanercept was rumoured to be used in horse racing in North America. To detect such use, the aim of this study was to develop a liquid chromatography-mass spectrometry (LC-MS) method for confirmation of etanercept in equine plasma. Etanercept was extracted from plasma by antihuman IgG antibody linked to magnetic beads. The analyte was reduced and alkylated, and then digested by trypsin. Tryptic peptides (T1 from human tumour necrosis factor receptor 2 of etanercept, T15 and T27 from human IgG1 of the protein) were employed for detection and confirmation of the analyte. The limit of detection was 5 ng/mL, and the limit of confirmation 10 ng/mL. This method is specific for confirmation of etanercept, as assessed using the results from BLAST and SEQUEST searches. The results from SEQUEST searches also revealed an unexpected unique specificity of product ion spectrum of IgG1 T27 with only a single product ion for identification of etanercept. It is the first report for such a finding, to the authors' knowledge. The method was successful in analyses of the plasma samples collected post administration of etanercept to horses. Etanercept was detected up to 11 days post administration. This method will be helpful for confirmation of etanercept or other protein-based drugs consisting of human IgG1, in equine drug testing.

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“…It has been noted in the literature that there is a strong preference for Pro to cleave at its N-terminal side, whether the product formed is the b ion or y ion. 30 Because b ions formed at most residues are accepted to have oxazolone structures (see Figure 1) [31][32][33] and because the b ion that would be formed at the C-terminal side of Pro would require a transition state involving formation of an unstable strained 5-5 bicyclic ring (see Figure 6), 6,8,34 cleavage typically occurs at the N-terminal side of Pro rather than the C-terminal side. This selective cleavage also provides information on the mechanism of the cleavage to form b and y ions.…”

Section: Nih-pa Author Manuscriptmentioning

confidence: 99%

Statistical Characterization of Ion Trap Tandem Mass Spectra from Doubly Charged Tryptic Peptides

et al. 2003

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Collision-induced dissociation (CID) is a common ion activation technique used to energize massselected peptide ions during tandem mass spectrometry. Characteristic fragment ions form from the cleavage of amide bonds within a peptide undergoing CID, allowing the inference of its amino acid sequence. The statistical characterization of these fragment ions is essential for improving peptide identification algorithms and for understanding the complex reactions taking place during CID. An examination of 1465 ion trap spectra from doubly charged tryptic peptides reveals several trends important to understanding this fragmentation process. While less abundant than y ions, b ions are present in sufficient numbers to aid sequencing algorithms. Fragment ions exhibit a characteristic series-specific relationship between their masses and intensities. Each residue influences fragmentation at adjacent amide bonds, with Pro quantifiably enhancing cleavage at its N-terminal amide bond and His increasing the formation of b ions at its C-terminal amide bond. Fragment ions corresponding to a formal loss of ammonia appear preferentially in peptides containing Gln and Asn. These trends are partially responsible for the complexity of peptide tandem mass spectra.Tandem mass spectrometry (MS/MS) of peptides is a central technology for proteomics, enabling the identification of thousands of peptides from a complex mixture. [1][2][3][4] This increasingly widespread technique relies upon the fragmentation of peptides by collisioninduced dissociation (CID), but the chemistry behind the fragmentation process is complex and not comprehensively understood. [5][6][7][8] Peptides undergo CID after they are isolated from other ions by their mass-to-charge (m/z) ratios. Peptides in an acidic solution are introduced to the vacuum of the mass spectrometer via electrospray ionization. 9 The peptide ions are accelerated during CID, leading to more energetic collisions with the ion trap's inert gas molecules. The mobile proton model 10 describes how the added internal energy causes the ionizing proton(s) on each peptide to transfer intramolecularly until one destabilizes a peptide bond, resulting in the cleavage of that bond and the production of two fragments. While more energetic techniques may cleave many classes of bonds within the peptide structure, low-energy CID preferentially breaks the amide bonds. Once the fragment ions are produced, the mass spectrometer records their m/z ratios in a tandem mass spectrum.Determining the sequence of a peptide from its tandem spectrum is complicated by the variety and variability of the fragment ions produced. Cleavage of amide bonds results in b and y Figure 1). b ions may fragment further to produce a ions. 13 If only these three ions were produced for every amide bond in a 10-residue peptide, the fragment ion spectrum would contain 27 peaks. This ideal spectrum differs from experimental spectra as a result of several causes. First, a subset of the expected fragment ions may not be present. Second...

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Probing Proline Effect in CID of Protonated Peptides

Cited by 170 publications

References 0 publications

Effect of the Identity of Xaa on the Fragmentation Modes of Doubly-Protonated Ala-Ala-Xaa-Ala-Ala-Ala-Arg

Effect of the Identity of Xaa on the Fragmentation Modes of Doubly-Protonated Ala-Ala-Xaa-Ala-Ala-Ala-Arg

Confirmatory analysis of etanercept in equine plasma by LC‐MS for doping control

Statistical Characterization of Ion Trap Tandem Mass Spectra from Doubly Charged Tryptic Peptides

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