N-Terminal amino acid side-chain cleavage of chemically modified peptides in the gas phase: A mass spectrometry technique for N-terminus identification

Chacon, Almary; Masterson, Douglas S.; Yin, Huiyong; Liebler, Daniel C.; Porter, Ned A.

doi:10.1016/j.bmc.2006.05.060

Cited by 27 publications

(35 citation statements)

References 18 publications

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“…The selective modification of lysine or N-terminus of a peptide is based on the peroxycarbamate chemistry reported recently [10,11]. Modification can be achieved by reaction of the peptide solution in ammonium bicarbonate buffer with t-butyl p-nitrophenyl peroxycarbonate.…”

Section: Resultsmentioning

confidence: 99%

“…The appearance of an immediate yellow color indicates the formation of p-nitrophenoxide. Even though Nterminus and lysine residues can be modified using the reagent, experimental conditions, especially the pH of the reaction medium, can be optimized to primarily modify lysine because the pK a for terminal ␣-amino group is about 8, whereas the -amino group of the lysine residue has a pK a of 10.7 [11]. The ion m/z 208 was generated in ionization source by increased spray voltage (source CID).…”

Section: Resultsmentioning

confidence: 99%

“…Series of fragments b 6 , c 6 are observed in the CID spectrum after lysine modification (Figure 3c). On the other hand, modification of the N-terminus by this peroxycarbonate method induces the same type of radical fragmentation, which can be used to identify the amino acid at the N-terminus [11]. Generally an imine fragment will be generated in this modification and the neutral loss of the side chain is amino acid dependent.…”

Section: Cid Experiments Of Modified Peptides At Lysine Sites Using Ementioning

confidence: 99%

“…Under the same modification conditions, the N-terminus can also be modified to some extent and the similar free-radical processes give rise to the loss of side chain of the amino acid at the N-terminus. The free-radical chemistry was recently explored to determine the N-terminus of peptides or proteins [11]. For example, in entry 1, the loss of side chain of valine at the N-terminus results in a fragment with m/z 961.45, whereas in entries 2, 3, and 5 side-chain loss of lysine, tyrosine, and serine are observed, respectively, in MALDI spectra.…”

Section: Modified Peptides Studied By Maldi Msmentioning

confidence: 99%

“…Fragmentation appears to result from initial free-radical dissociation of the peroxide bond followed by decarboxylation. The chemistry can also be used for N-terminus amino acid identification based on the neutral loss of the side chain [11]. The "digestion" of peptides or proteins in the gas phase after chemical derivatization may provide an alternative to enzyme digestion and could potentially increase the throughput of proteomics analysis.…”

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confidence: 99%

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Free radical-induced site-specific peptide cleavage in the gas phase: Low-energy collision-induced dissociation in ESI- and MALDI mass spectrometry

Yin

Chacon

Porter

et al. 2007

J. Am. Soc. Mass Spectrom.

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Protein identification is routinely accomplished by peptide sequencing using mass spectrometry (MS) after enzymatic digestion. Site-specific chemical modification may improve peptide ionization efficiency or sequence coverage in mass spectrometry. We report herein that amino group of lysine residue in peptides can be selectively modified by reaction with a peroxycarbonate and the resulting lysine peroxycarbamates undergo homolytic fragmentation under conditions of low-energy collision-induced dissociation (CID) in electrospray ionization (ESI) and matrix-assisted laser desorption and ionization (MALDI) MS. Selective modification of lysine residue in peptides by our strategy can induce specific peptide cleavage at or near the lysine site. Studies using deuterated analogues of modified lysine indicate that fragmentation of the modified peptides involves apparent free-radical processes that lead to peptide chain fragmentation and side-chain loss. The formation of a-, c-, or z-types of ions in MS is reminiscent of the proposed free-radical mechanisms in low-energy electron capture dissociation (ECD) processes that may have better sequence coverage than that of the conventional CID method. This site-specific cleavage of peptides by free radical-promoted processes is feasible and such strategies may aid the protein sequencing analysis and have potential applications in top-down proteomics. . The sequencing is often accomplished using tandem mass spectrometry (MS/MS) by collision-induced dissociation (CID) or electron capture dissociation (ECD) of protonated species [3,4]. Chemical modification of peptides or proteins has also provided strategies that are helpful in assignment of peptide sequence, enhancement of the MS sensitivity [5], and, importantly, in protein quantification [6]. Even though database searches for protein identification are primarily based on b-and y-ions observed in MS/MS spectra, complex fragmentation patterns can result from the ECD process and improve the sequence coverage in protein identification [7,8]. The typical a-, c-, and z-fragments in ECD are postulated to arise from free-radical intermediates. The nomenclature of the fragments as proposed by Roestorff is shown in Scheme 1 [9].In a recent communication we reported a method to generate radicals in a site-specific manner on peptides in the gas phase, after modification of lysine residues as peroxycarbamates [10]. Fragmentation appears to result from initial free-radical dissociation of the peroxide bond followed by decarboxylation. The chemistry can

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Cid Experiments Of Modified Peptides At Lysine Sites Using Ementioning

confidence: 99%

Section: Modified Peptides Studied By Maldi Msmentioning

confidence: 99%

mentioning

confidence: 99%

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Free radical-induced site-specific peptide cleavage in the gas phase: Low-energy collision-induced dissociation in ESI- and MALDI mass spectrometry

Yin

Chacon

Porter

et al. 2007

J. Am. Soc. Mass Spectrom.

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Structure and Reactivity of the Glutathione Radical Cation: Radical Rearrangement from the Cysteine Sulfur to the Glutamic Acid α‐Carbon Atom

et al. 2013

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A gas‐phase radical rearrangement through intramolecular hydrogen‐atom transfer (HAT) was studied in the glutathione radical cation, [γ‐ECG]+., which was generated by a homolytic cleavage of the protonated S‐nitrosoglutathione. Ion–molecule reactions suggested that the radical migrates from the original sulfur position to one of the α‐carbon atoms. Experiments on the radical cations of dipeptides derived from the glutathione sequence, [γ‐EC]+. and [CG]+., pointed to the glutamic acid α‐carbon atom as the most likely site of the radical migration. Infrared multiple‐photon dissociation (IRMPD) spectroscopy was employed to generate complementary information. IRMPD of [γ‐ECG]+. in the approximately 1000–1800 cm−1 region was inconclusive owing to the relatively broad, overlapping absorption bands. However, the IRMPD spectrum of [γ‐EC]+. in this region was consistent with the radical migrating from the sulfur to the α‐carbon atom of glutamic acid. IRMPD in the 2800–3700 cm−1 region performed on [γ‐ECG]+. is consistent with a mixture of both the original sulfur‐based radical and the resulting glutamic acid α‐carbon‐based species. Comparisons are made with previously published condensed and gas‐phase studies on intramolecular HAT in glutathione.

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Gas‐phase peptide fragmentation: how understanding the fundamentals provides a springboard to developing new chemistry and novel proteomic tools

Barlow

O’Hair

2008

J. Mass Spectrom.

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This tutorial provides an overview of the evolution of some of the key concepts in the gas-phase fragmentation of different classes of peptide ions under various conditions [e.g. collision-induced dissociation (CID) and electron transfer dissociation (ETD)], and then demonstrates how these concepts can be used to develop new methods. For example, an understanding of the role of the mobile proton and neighboring group interactions in the fragmentation reactions of protonated peptides has led to the design of the 'SELECT' method. For ETD, a model based on the Landau-Zener theory reveals the role of both thermodynamic and geometric effects in the electron transfer from polyatomic reagent anions to multiply protonated peptides, and this predictive model has facilitated the design of a new strategy to form ETD reagent anions from precursors generated via ESI. Finally, two promising, emerging areas of gas-phase ion chemistry of peptides are also described: (1) the design of new gas-phase radical chemistry to probe peptide structure, and (2) selective cleavage of disulfide bonds of peptides in the gas phase via various physicochemical approaches.

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N-Terminal amino acid side-chain cleavage of chemically modified peptides in the gas phase: A mass spectrometry technique for N-terminus identification

Cited by 27 publications

References 18 publications

Free radical-induced site-specific peptide cleavage in the gas phase: Low-energy collision-induced dissociation in ESI- and MALDI mass spectrometry

Free radical-induced site-specific peptide cleavage in the gas phase: Low-energy collision-induced dissociation in ESI- and MALDI mass spectrometry

Structure and Reactivity of the Glutathione Radical Cation: Radical Rearrangement from the Cysteine Sulfur to the Glutamic Acid α‐Carbon Atom

Gas‐phase peptide fragmentation: how understanding the fundamentals provides a springboard to developing new chemistry and novel proteomic tools

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