[22] Matrix-assisted laser desorption ionization mass-spectrometry of proteins

Beavis, Ronald C.; Chait, Brian T.

doi:10.1016/s0076-6879(96)70024-1

Cited by 157 publications

(102 citation statements)

References 60 publications

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“…The breakdown graph for MH ϩ obtained by cone-voltage CID is presented in Figure 4. Clearly, formation of b 4 is the most prominent fragmentation reaction with minor formation of y 3 , b 3 , b 5 (-H 2 O), and y 2 . We believe that the apparent higher onset for the y 2 product in Figure 4 is not real but reflects the detection limit for low-intensity ions.…”

Section: Tetraalaninementioning

confidence: 99%

“…W ith the advent of soft ionization techniques such as electrospray ionization (ESI) [1][2][3] and matrix-assisted laser desorption-ionization (MALDI) [4,5] which efficiently ionize by protonation a wide variety of peptides and proteins, tandem mass spectrometry [6,7] has become a method of significant importance for the sequencing of peptides through collision-induced dissociation (CID) studies. As a result, the main types of fragmentation reactions occurring are well-established [8 -11], at least in a phenomenological sense and are outlined in Scheme 1.…”

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confidence: 99%

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Fragmentation of protonated oligoalanines: Amide bond cleavage and beyond

Harrison

Young

2004

J. Am. Soc. Mass Spectrom.

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The fragmentation reactions of the singly-protonated oligoalanines trialanine to hexaalanine have been studied using energy-resolved mass spectrometry in MS 2 and MS 3 experiments. The primary fragmentation reactions are rationalized in terms of the b x -y z pathway of amide bond cleavage which results in formation of a proton-bound complex of an oxazolone and a peptide/amino acid; on decomposition of this complex the species of higher proton affinity preferentially retains the proton. For protonated pentaalanine and protonated hexaalanine the major primary fragmentation reaction involves cleavage of the C-terminal amide bond to form the appropriate b ion. The lower mass b ions originate largely, if not completely, by further fragmentation of the initially formed b ion. MS 3 energy-resolved experiments clearly show the fragmentation sequence b n 3 b nϪ1 3 b nϪ2 . A more minor pathway for the alanines involves the sequence b n 3 a n 3 b nϪ1 3 b nϪ2 . The a 5 ion formed from hexaalanine loses, in part, NH 3 to begin the sequence of fragmentation reactions a 5 3 a 5 * 3 a 4 * 3 a 3 * where a n * ϭ a n Ϫ NH 3 . The a 3 * ion also is formed from the b 3 ion by the sequence b 3 3 a 3 3 a 3 * with the final step being sufficiently facile that the a 3 ion is not observed with significant intensity in CID mass spectra. and matrix-assisted laser desorption-ionization (MALDI) [4,5] which efficiently ionize by protonation a wide variety of peptides and proteins, tandem mass spectrometry [6,7] has become a method of significant importance for the sequencing of peptides through collision-induced dissociation (CID) studies. As a result, the main types of fragmentation reactions occurring are well-established [8 -11], at least in a phenomenological sense and are outlined in Scheme 1. A major mode of fragmentation in many cases involves cleavage of an amide bond in the protonated peptide. When the charge is retained by the C-terminus fragment migration of a labile hydrogen from the Nterminus neutral fragment occurs to form a protonated amino acid (y 1 ) or peptide (y n ) [12,13]. When the charge remains on the N-terminus fragment, a neutral amino acid or peptide is eliminated and b n ions are formed. Although these b n ions were initially considered to be acylium ions [8,10,11], extensive experimental and theoretical studies [14 -19] have shown that, in many cases, the b n ions have protonated oxazolone structure as shown in Scheme 2 formed by cyclization involving the carbonyl function next-nearest to the amide bond being ruptured. In several cases where there are more reactive side-chain groups in the peptide, alternative cyclic structures are formed by interaction with sidechain functionality as the amide bond cleavage occurs [20,21].Although the fragmentation reactions of b 2 ions have been studied in some detail [14,18,22,23], the fragmentation reactions of larger b ions have seen less study [15] and the fragmentation of a n ions have seen even less study. A related question is whether, for larger protonated peptides, the ...

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Section: Tetraalaninementioning

confidence: 99%

mentioning

confidence: 99%

Fragmentation of protonated oligoalanines: Amide bond cleavage and beyond

Harrison

Young

2004

J. Am. Soc. Mass Spectrom.

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“…Aliquots of protein in a series of 1:2 dilutions were mixed with matrix solution and then spotted onto the sample plate using the sandwich method (23). MALDI-TOF spectrometric data were collected using a Voyager Biospectrotometry work station, operated in linear mode.…”

Section: Cloning and Expression Plasmid Construct-mentioning

confidence: 99%

Kinetic Study of the Antiport Mechanism of an Escherichia coli Zinc Transporter, ZitB

Chao

2004

Journal of Biological Chemistry

153

140

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ZitB is a member of the cation diffusion facilitator (CDF) family that mediates efflux of zinc across the plasma membrane of Escherichia coli. We describe the first kinetic study of the purified and reconstituted ZitB by stopped-flow measurements of transmembrane fluxes of metal ions using a metal-sensitive fluorescent indicator encapsulated in proteoliposomes. Metal ion filling experiments showed that the initial rate of Zn 2؉ influx was a linear function of the molar ratio of ZitB to lipid and was related to the concentration of Zn 2؉ or Cd 2؉ by a hyperbola with a Michaelis-Menten constant (K m ) of 104.9 ؎ 5.4 M and 90.1 ؎ 3.7 M, respectively. Depletion of proton stalled Cd 2؉ transport down its diffusion gradient, whereas tetraethylammonium ion substitution for K ؉ did not affect Cd 2؉ transport, indicating that Cd 2؉ transport is coupled to H ؉ rather than to K ؉ . H ؉ transport was inferred by the H ؉ dependence of Cd 2؉ transport, showing a hyperbolic relationship with a K m of 19.9 nM for H ؉ . Applying H ؉ diffusion gradients across the membrane caused Cd 2؉ fluxes both into and out of proteoliposomes against the imposed H ؉ gradients. Likewise, applying outwardly oriented membrane electrical potential resulted in Cd 2؉ efflux, demonstrating the electrogenic effect of ZitB transport. Taken together, these results indicate that ZitB is an antiporter catalyzing the obligatory exchange of Zn 2؉ or Cd 2؉ for H ؉ . The exchange stoichiometry of metal ion for proton is likely to be 1:1.

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“…The last is formed by cleavage of the His-Gly amide bond on a pathway that is initiated by nucleophilic attack of one of the His side-chain imidazole nitrogens. [1][2][3] and matrix-assisted laser desorption ionization (MALDI) [4,5], tandem mass spectrometry [6,7] of protonated or multiply-protonated peptides has become the method of choice for identifying the amino acid sequence of peptides. As a result of many studies, the main fragmentation channels of singly-protonated peptides have been established, and considerable progress has been made in understanding, in detail, the mechanistic aspects of the fragmentation reactions; these studies have been the subject of several recent reviews [8 -13].…”

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confidence: 99%

Fragmentation of doubly-protonated Pro-His-Xaa tripeptides: Formation of b₂²⁺ ions

Knapp-Mohammady

Young

Paizs

et al. 2009

J. Am. Soc. Mass Spectrom.

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When ionized by electrospray from acidic solutions, the tripeptides Pro-His-Xaa (Xaa ϭ Gly, Ala, Leu) form abundant doubly-protonated ions, [M ϩ 2H] 2ϩ . Collision-induced dissociation (CID) of these doubly-protonated species results, in part, in formation of b 2 2ϩ ions, which fragment further by loss of CO to form a 2 2ϩ ions; the latter fragment by loss of CO to form the Pro and His iminium [immonium is commonly used in peptide MS work] ions. Although larger doubly-charged b ions are known, this represents the first detailed study of b 2 2ϩ ions in CID of small doubly protonated peptides. The most abundant CID products of the studied doubly-protonated peptides arise mainly in charge separation involving two primary fragmentation channels, formation of the b 2 /y 1 pair and formation of the a 1 /y 2 pair. Combined molecular dynamics and density functional theory calculations are used to gain insight into the structures and fragmentation pathways of doubly-protonated Pro-His-Gly including the energetics of potential protonation sites, backbone cleavages, post-cleavage charge-separation reactions and the isomeric structures of b 2 2ϩ ions. Three possible structures are considered for the b 2 2ϩ ions: the oxazolone, diketopiperazine, and fused ring isomers. The last is formed by cleavage of the His-Gly amide bond on a pathway that is initiated by nucleophilic attack of one of the His side-chain imidazole nitrogens. Our calculations indicate the b 2 2ϩ ion population is dominated by the oxazolone and/or fused ring isomers. (J Am Soc Mass Spectrom 2009, 20, 2135-2143

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[22] Matrix-assisted laser desorption ionization mass-spectrometry of proteins

Cited by 157 publications

References 60 publications

Fragmentation of protonated oligoalanines: Amide bond cleavage and beyond

Fragmentation of protonated oligoalanines: Amide bond cleavage and beyond

Kinetic Study of the Antiport Mechanism of an Escherichia coli Zinc Transporter, ZitB

Fragmentation of doubly-protonated Pro-His-Xaa tripeptides: Formation of b₂²⁺ ions

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[22] Matrix-assisted laser desorption ionization mass-spectrometry of proteins

Cited by 157 publications

References 60 publications

Fragmentation of protonated oligoalanines: Amide bond cleavage and beyond

Fragmentation of protonated oligoalanines: Amide bond cleavage and beyond

Kinetic Study of the Antiport Mechanism of an Escherichia coli Zinc Transporter, ZitB

Fragmentation of doubly-protonated Pro-His-Xaa tripeptides: Formation of b22+ ions

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Fragmentation of doubly-protonated Pro-His-Xaa tripeptides: Formation of b₂²⁺ ions