An unusual cleavage reaction of a peptide observed during dithiotreitol and tris(2-carboxyethyl)phosphine reduction: application to sequencing of HpTx2 spider toxin using nanospray tandem mass spectrometry

A recombinant peptidic spider toxin, HpTx2, was investigated directly by nanoelectrospray tandem mass spectrometry (MS/MS). This 30-residue toxin possesses a highly knotted structure with cystines arranged in close proximity. The low-energy collision-induced dissociation MS/MS spectrum of the [M+4H](4+) ion permitted characterization of the C-terminal sequence of HpTx2 up to Cys(26) that is involved in a disulfide bridge. Chemical pre-treatment with DTT or TCEP was then investigated, and it was found that an unexpected cleavage reaction of HpTx2 gave two smaller peptides which were completely sequenced by MS/MS experiments using a Qq-TOF mass spectrometer. This unusual hydrolysis reaction facilitated the determination of the complete sequence of the HpTx2 toxin.     

Disulfide bond cleavages observed in SORI-CID of three nonapeptides complexed with divalent transition-metal cations

Tandem MS sequencing of peptides that contain a disulfide bond is often hampered when using a slow heating technique. We show that complexation of a transition-metal ion with a disulfide-bridge-containing nonapeptide yields very rich tandem mass spectra, including fragments that involve the cleavage of the disulfide bond up to 56% of the total product ion intensity. On the contrary, MS/MS of the corresponding protonated nonapeptides results predominantly in fragments from the region that is not involved in the disulfide bond. Eleven different combinations of three nonapeptides and three metal ions were measured using Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) combined with sustained off-resonance irradiation collision induced dissociation (SORI-CID). All observed fragments are discussed with respect to four different types of product ions: neutral losses, b/y-fragmentation with and without the disulfide bond cleavage, and losses of internal amino acids without rupture of the disulfide bridge. Furthermore, it is shown that the observed complementary fragment pairs obtained from peptide-metal complexes can be used to determine the region of the binding site of the metal ion. This approach offers an efficient way to cleave disulfide-bridged structures using low energy MS/MS, which leads to increased sequence coverage and more confidence in peptide or protein assignments.     

Fragmentation studies of peptides: the formation of y ions.

The mechanism for the formation of y ions in the collision-induced dissociation (CID) spectra of protonated peptides produced by fast-atom bombardment was investigated by tandem mass spectrometry and deuterium labelling studies. The results show that a hydrogen atom attached to nitrogen and not to carbon migrates during cleavage of the amide bond. A mechanism based on these results is presented.     

Does Electron Capture Dissociation Cleave Protein Disulfide Bonds?

Peptide and protein characterization by mass spectrometry (MS) relies on their dissociation in the gas phase into specific fragments whose mass values can be aligned as ‘mass ladders’ to provide sequence information and to localize possible posttranslational modifications. The most common dissociation method involves slow heating of even-electron (M+n H)ⁿ⁺ ions from electrospray ionization by energetic collisions with inert gas, and cleavage of amide backbone bonds. More recently, dissociation methods based on electron capture or transfer were found to provide far more extensive sequence coverage through unselective cleavage of backbone N–Cα bonds. As another important feature of electron capture dissociation (ECD) and electron transfer dissociation (ETD), their unique unimolecular radical ion chemistry generally preserves labile posttranslational modifications such as glycosylation and phosphorylation. Moreover, it was postulated that disulfide bond cleavage is preferred over backbone cleavage, and that capture of a single electron can break both a backbone and a disulfide bond, or even two disulfide bonds between two peptide chains. However, the proposal of preferential disulfide bond cleavage in ECD or ETD has recently been debated. The experimental data presented here reveal that the mechanism of protein disulfide bond cleavage is much more intricate than previously anticipated.     

Dissociation of disulfide‐intact somatostatin ions: the roles of ion type and dissociation method

The dissociation chemistry of somatostatin‐14 was examined using various tandem mass spectrometry techniques including low‐energy beam‐type and ion trap collision‐induced dissociation (CID) of protonated and deprotonated forms of the peptide, CID of peptide‐gold complexes, and electron transfer dissociation (ETD) of cations. Most of the sequence of somatostatin‐14 is present within a loop defined by the disulfide linkage between Cys‐3 and Cys‐14. The generation of readily interpretable sequence‐related ions from within the loop requires the cleavage of at least one of the bonds of the disulfide linkage and the cleavage of one polypeptide backbone bond. CID of the protonated forms of somatostatin did not appear to give rise to an appreciable degree of dissociation of the disulfide linkage. Sequential fragmentation via multiple alternative pathways tended to generate very complex spectra. CID of the anions proceeded through CH₂? S cleavages extensively but relatively few structurally diagnostic ions were generated. The incorporation of Au(I) into the molecule via ion/ion reactions followed by CID gave rise to many structurally relevant dissociation products, particularly for the [M+Au+H]²⁺ species. The products were generated by a combination of S? S bond cleavage and amide bond cleavage. ETD of the [M+3H]³⁺ ion generated rich sequence information, as did CID of the electron transfer products that did not fragment directly upon electron transfer. The electron transfer results suggest that both the S? S bond and an N? C_α bond can be cleaved following a single electron transfer reaction. Copyright © 2009 John Wiley & Sons, Ltd.     

Cleavage of Multiple Disulfide Bonds in Insulin via Gold Cationization and Collision-induced Dissociation

Intact bovine insulin, with its two chains linked via two disulfide linkages, has been used as a model system to study the incorporation of one or more gold cations as means for facilitating the cleavage of multiple disulfide bonds in a tandem mass spectrometry experiment. Gas-phase ion/ion reactions involving Au(I)Cl(2) (-) or Au(III)Cl(4) (-) were used to incorporate either one or two gold cations into multiply-protonated insulin cations, followed by ion trap collision-induced dissociation (CID) of the products. The incorporation of a single gold cation followed by CID showed little evidence for disulfide bond cleavage. Rather, the CID spectra were similar to those acquired for the same charge state with only excess protons present. However, the incorporation of two gold cations, regardless of oxidation state, resulted in efficient cleavage of the disulfide bonds connecting the two chains of insulin. Furthermore, ion trap CID of the insulin complexes containing two gold cations showed more sequence information compared to the complexes containing only one gold cation or no gold cations. The partitioning of the gold cations between the two chains upon CID proved to be largely asymmetric, as both gold cations tended to stay together. There appeared to be a slight preference for both gold cations to partition into the B-chain. However, the relatively low contribution from single chain ions with only one gold ion suggests a degree of cooperativity in the overall mechanism for separation of the two chains.     

Fragmentation of intra-peptide and inter-peptide disulfide bonds of proteolytic peptides by nanoESI collision-induced dissociation

Characterisation and identification of disulfide bridges is an important aspect of structural elucidation of proteins. Covalent cysteine-cysteine contacts within the protein give rise to stabilisation of the native tertiary structure of the molecules. Bottom-up identification and sequencing of proteins by mass spectrometry most frequently involves reductive cleavage and alkylation of disulfide links followed by enzymatic digestion. However, when using this approach, information on cysteine-cysteine contacts within the protein is lost. Mass spectrometric characterisation of peptides containing intra-chain disulfides is a challenging analytical task, because peptide bonds within the disulfide loop are believed to be resistant to fragmentation. In this contribution we show recent results on the fragmentation of intra and inter-peptide disulfide bonds of proteolytic peptides by nano electrospray ionisation collision-induced dissociation (nanoESI CID). Disulfide bridge-containing peptides obtained from proteolytic digests were submitted to low-energy nanoESI CID using a quadrupole time-of-flight (Q-TOF) instrument as a mass analyser. Fragmentation of the gaseous peptide ions gave rise to a set of b and y-type fragment ions which enabled derivation of the sequence of the amino acids located outside the disulfide loop. Surprisingly, careful examination of the fragment-ion spectra of peptide ions comprising an intramolecular disulfide bridge revealed the presence of low-abundance fragment ions formed by the cleavage of peptide bonds within the disulfide loop. These fragmentations are preceded by proton-induced asymmetric cleavage of the disulfide bridge giving rise to a modified cysteine containing a disulfohydryl substituent and a dehydroalanine residue on the C-S cleavage site.     

Mass spectrometric characterization of transferrins and their fragments derived by reduction of disulfide bonds

Mass spectrometry, proteomics, and protein chemistry methods are used to characterize the cleavage products of 79 kDa transferrin proteins induced by iron-catalyzed oxidation, including a novel C-terminal polypeptide released upon disulfide reduction. Top-down electrospray ionization tandem mass spectrometry (ESI-MS/MS) of intact multiply-charged transferrin from a variety of species (human, bovine, rabbit, chicken) performed on a quadrupole time-of-flight mass spectrometer yields multiply-charged b(n)-products originating near residues 56-69 from the N-terminal region, in addition to their complementary y(n)-products. Incubation of transferrin with reductants, such as dithiothreitol (DTT) or tris(2-carboxyethyl)-phosphine (TCEP), yields an increase in multiple charging observed by ESI-MS and an increase in molecular weight consistent with disulfide reduction. However, mammalian transferrins release a 6-8 kDa fragment upon disulfide reduction. Protein acetylation and MS/MS sequencing demonstrate that the fragment originates from the C-terminus of the protein, and that it is a separate polypeptide linked via three disulfide bonds to the main transferrin chain. The existence of a separate C-terminal chain is not annotated in protein sequence databases and, to date, has not been reported in the literature. Iron-catalyzed cleavage induces fragments originating from both the N- and C-terminus of transferrin.     

Application of MALDI TOF/TOF mass spectrometry and collision-induced dissociation for the identification of disulfide-bonded peptides

This paper describes a method for the fast identification and composition of disulfide-bonded peptides. A unique fragmentation signature of inter-disulfide-bonded peptides is detected using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF)/TOF mass spectrometry and high-energy collision-induced dissociation (CID). This fragmentation pattern identifies peptides with an interconnected disulfide bond and provides information regarding the composition of the peptides involved in the pairing. The distinctive signature produced using CID is a triplet of ions resulting from the cleavage of the disulfide bond to produce dehydroalanine, cysteine or thiocysteine product ions. This method is not applicable to intra-peptide disulfide bonds, as the cleavage mechanism is not the same and a triplet pattern is not observed. This method has been successfully applied to identifying disulfide-bonded peptides in a number of control digestions, as well as study samples where disulfide bond networks were postulated and/or unknown.     

化学裂解结合生物质谱对多肽二硫键的定位

二硫键是一种与多肽及蛋白质结构和功能密切相关的化学键.当多肽中存在多个半胱氨酸时,形成的二硫键可能会存在多种配对方式.快速且精准地定位多肽中多对二硫键对研究多肽的结构与功能间的关系十分重要.本文开发了一种基于化学裂解和生物质谱的新方法,对利那洛肽中3对二硫键进行了精准定位.通过解析裂解后特异肽段的二级质谱图,确定利那洛肽中3对二硫键的配对方式分别为Cys1-Cys6,Cys2-Cys10和Cys5-Cys13.该方法为二硫键的定位研究提供了新思路.     

Electron Capture Dissociation of Disulfide, Sulfur–Selenium, and Diselenide Bound Peptides

To examine the electron capture dissociation (ECD) behavior of disulfide (S?CS), sulfur?Cselenium (S?CSe), and diselenide (Se?CSe) bonds-containing peptides, a series of free cysteine (Cys) and selenocysteine (Sec) containing peptides were reacted to form interchain S?CS, S?CSe, and Se?CSe bonds, and then studied using ECD with Fourier transform ion cyclotron mass spectrometry (FTICR MS). These results demonstrate that the radical has higher tendency to stay at selenium rather than sulfur after the cleavage of Se?CS bonds by ECD. In addition, ?CSH (?C33), ?CS (?C32), and ?CS + H (?C31) small neutral losses were all observed from the cleavage of C?CS bonds of a disulfide bound peptide. Similar, but minor, fragments were also detected in S?CSe bound peptides. In contrast, the cleavage of C?CSe bonds of the Se?CSe species mainly forms fragments with neutral loss of ?CSe + H (?C78.90868), and the radical tends to stay on the selenium of its corresponding complementary pair. Although the electron affinities of S atom (2.07?eV) and Se atom (2.02?eV) are very close; they have very different reactivity towards electrons. The replacement of sulfur with selenium greatly increases the electron affinities of S?CSe and Se?CSe bonds comparing to S?CS bonds (with an increase of electron affinity by about 0.20?eV by replacing a sulfur with a selenium) (Int J Quantum Chem 110:513-523, 2010), which in turn leads to different ECD fragmentation behavior and mechanisms. Our results are in good agreement with previously published ab initio calculations on Se?CSe compounds by other groups.     

Fragmentation of peptide disulfides under conditions of negative ion mass spectrometry: Studies of oxidized glutathione and contryphan

The fragmentation of positive and negative ions of peptide disulfides under mass spectrometric conditions yields distinctly different product ion distributions. A negative ion upon collision induced dissociation yields intense product ions, which correspond to cleavage at the disulfide linkage. The complete assignment of the product ions obtained upon fragmentation of oxidized glutathione in an ion trap is presented. The cleavage at the disulfide site is mediated by abstraction of CalphaH and CbetaH protons resulting in product ions derived by neutral loss of H2S2 and H2S. The formation of peptide thioaldehydes and persulfides at the cysteine sites is established. Dehydroalanine formation at the Cys residue is predominant. The case of a contryphan, a cyclic peptide disulfide derived from Conus snail venom, illustrates the utility of negative ion mass spectrometry in disulfide identification. Complementary information is derived by combining the fragmentation patterns obtained from positive and negative ions of disulfide containing peptides.     

Application of ion trap technology to liquid chromatography/mass spectrometry quantitation of large peptides

Triple quadrupole mass spectrometers are generally considered the instrument of choice for quantitative analysis. However, for the analysis of large peptides we have encountered some cases where, as the data presented here would indicate, ion trap mass spectrometers may be a good alternative. In general, specificity and sensitivity in bioanalytical liquid chromatography/mass spectrometry (LC/MS) assays are achieved via tandem MS (MS/MS) utilizing collision-induced dissociation (CID) while monitoring unique precursor to product ion transitions (i.e. selected reaction monitoring, SRM). Due to the difference in CID processes, triple quadrupoles and ion traps often generate significantly different fragmentation spectra of product ion species and intensities. The large peptidic analytes investigated here generated fewer fragments with higher relative abundance on the ion trap as compared to those generated on the triple quadrupole, resulting in lower limits of detection on the ion trap.     

Top‐down proteomics with a quadrupole time‐of‐flight mass spectrometer and collision‐induced dissociation

With slight modifications of the instrumental parameters, we demonstrate that satisfactory top‐down data can be obtained with collision‐induced dissociation (CID) tandem mass spectrometry on a quadrupole time‐of‐flight (qTOF) instrument not originally designed for this purpose. Protein identification is achieved with both N‐ and C‐terminal sequence tags and BLAST database searches. The accurate mass measurement of multiply charged fragment ions (mostly y and b‐type) supplements the limited set of cleavage sites and provides a high degree of sequence coverage (90–100%). Post‐translational modification issues can be addressed too. This approach might help those mass spectrometry (MS) core facilities that are not able to afford very high‐resolution instruments, thus expanding the benefits of top‐down protein analysis over the worldwide MS community. Copyright © 2009 John Wiley & Sons, Ltd.     

Can metal ions be used as gas‐phase disulfide bond cleavage reagents? A survey of coinage metal complexes of model peptides containing an intermolecular disulfide bond

The role that a metal ion can have in promoting disulfide bond cleavage has been assessed by surveying the tandem mass spectra of the following metal complexes of model peptides containing an intermolecular disulfide bond: [M--H+Cu(II)](+); [M--H+Cu(II)(bipy)](+); [M+Ag(I)](+); and [M+Au(I)(PMe(3))](+). In comparison to previously studied protonated peptides, these binary and ternary metal complexes generally yield more abundant S--S and/or C--S bond cleavage. In general, [M--H+Cu(II)](+) ions cleave the adjacent C--S bond more readily, while the [M+Au(I)(PMe(3))](+) ion cleaves the S--S bond more readily. The ternary metal complex [M--H+Cu(II)(bipy)](+), on the other hand, fragments by exclusive loss of the bipyridyl ligand for the larger model peptides studied. Of all coinage metal systems studied, Me(3)PAu(+) is superior in promoting disulfide bond cleavage.     

Concerning the gas chromatographic determination of amphetamine as (1-phenyl-isopropyl)-isothiocyanate

Amphetamine (I) reacts spontaneously at room temperature with carbon disulfide to form (1-phenyl-isopropyl)-isothiocyanate, which was identified by UV.-, IR.- and mass spectrometry. This reaction is used for the analytical determination of I by gas chromatography. The characteristic peak shift which occurs after addition of carbon disulfide differentiates I from N-methylamphetamine (II). The method is applied to urine analysis of I and II (which partially metabolizes to I) and might be useful in doping control.     

Adsorption Properties of Thiocontaining Schungite

Chromatography–mass spectrometry and thermodesorption mass spectrometry have been employed to study mineral schungite-III, in which various thiocompounds have been detected and identified. The influence of these compounds on the adsorption activity of schungite with respect to iodine has been investigated. It has been shown that sulfur present in schungite has no effect on the results of determining its adsorption activity and does not interact with iodine; however, it can interact with amino compounds to yield sulfides. Activation energies E_a have been experimentally determined for sulfur, iodine, and dimethyl disulfide desorption from the surfaces of schungite and a model sorbent, graphitized thermal carbon black. The E_a values of these compounds have appeared to be several times lower than the heats of their adsorption on carbon black calculated by the molecular-statistical method.     

Comparison of supercritical fluid chromatography–tandem mass spectrometry and liquid chromatography–tandem mass spectrometry for the stereoselective analysis of chlorfenvinphos and dimethylvinphos in tobacco

This study used reversed-phase liquid chromatography–tandem mass spectrometry and supercritical fluid chromatography–tandem mass spectrometry for determination of the stereoisomers of chlorfenvinphos and dimethylvinphos in tobacco. Tobacco samples were extracted and purified with a modified quick, easy, cheap, effective, rugged, and safe technique using spherical carbon. The performance of both methodologies was comprehensively compared in terms of methods validation parameters (separation efficiency, linearity, selectivity, recovery, repeatability, sensitivity, matrix effect, etc.). Under optimized conditions, the calibration curves of the stereoisomers of chlorfenvinphos and dimethylvinphos in the range of 10–500 ng/mL showed excellent linearity with R² ≥ 0.997 in both methods. The adequate recoveries of analytes from three different spiked tobaccos were obtained using reversed-phase liquid chromatography–tandem mass spectrometry (86.1–95.7%) as well as supercritical fluid chromatography–tandem mass spectrometry (86.5–94.0%). The relative standard deviations for spiked samples were all below 7.0%. Compared with supercritical fluid chromatography–tandem mass spectrometry, lower matrix effects and LODs can be obtained in reversed-phase liquid chromatography–tandem mass spectrometry.     

An investigation of the homolytic saccharide cleavage of deprotonated flavonol 3-O-glycosides in a quadrupole ion trap mass spectrometer

The trend in the extent of homolytic saccharide cleavage is reported for a series of deprotonated flavonol 3-O-glycosides upon collision-induced dissociation (CID) in a quadrupole ion trap mass spectrometer. The second-generation product ions from the primary [Y(0)](-) and [Y(0)- H](-.) product ions were also identified. It was determined that the structure of both the aglycon and the saccharide portions of the flavonoid glycoside are pivotal in inducing radical cleavage. In contrast to earlier work on this subject reported for a smaller group of flavonols, the correlation between the degree of B-ring hydroxylation and the extent of radical saccharide cleavage showed several notable exceptions in the present work. Homolytic cleavage was also investigated in the context of using tandem mass spectrometry to identify the aglycon portions of flavonoid glycosides.     

Application of electrospray ionization with Fourier transform ion cyclotron resonance mass spectrometry for structural screening of core oligosaccharides from lipopolysaccharides of the bacteria Proteus

Electrospray ionization with Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICRMS) was used for screening and structural elucidation of core oligosaccharides isolated from lipopolysaccharides of bacteria of the genus Proteus. Mass spectra allowed the determination of the molecular masses with high accuracy and the estimation of the chemical heterogeneity of the samples. They did not, however, provide sufficient information to identify structural details of the branched oligosaccharides. Therefore, various fragmentation techniques for determining such details were examined. Infrared multiphoton dissociation tandem mass spectrometry (IRMPD-MS/MS) experiments in negative ion mode resulted in cleavage between the structurally conserved inner core region and the variable outer core region. Positive ion capillary skimmer dissociation mass spectra showed numerous fragment ion peaks, including those corresponding to the subsequent cleavage of the glycosidic linkages starting from the non-reducing end of the oligosaccharide. Despite their complexity, these mass spectrometric studies allowed confirmation of previously determined Proteus lipopolysaccharide core structures, and identification of new related structures in other strains of these bacteria.