Side-chain Cleavages Research Articles

The Effect of Radical Trap Moieties on Electron Capture Dissociation Spectra of Substance P

To further test the hypothesis that electron capture dissociation (ECD) involves long-lived radical intermediates and radical migration occurs within these intermediates before fragmentation, radical trap moieties were attached to peptides with the assumption that they would reduce fragmentation by decreasing the mobility of the radical. Coumarin labels were chosen for the radical traps, and unlabeled, singly-labeled, and doubly-labeled Substance P were analyzed by ECD. The results demonstrated a correlation between the number and position of tags on the peptide and the intensity of side-chain cleavages observed, as well as an inverse correlation between the number of tags on the peptide and the intensity of backbone cleavages. Addition of radical traps to the peptide inhibits backbone cleavages, suggesting that either radical mobility is required for these cleavages, or new noncovalent interactions prevent separation of backbone cleavage fragments. The enhancement of side-chain cleavages and the observation of new side-chain cleavages associated with aromatic groups suggest that the gas-phase conformation of this peptide is substantially distorted from untagged Substance P and involves previously unobserved interactions between the coumarin tags and the phenylalanine residues. Furthermore, the use of a double resonance (DR)-ECD experiment showed that these side-chain losses are all products of long-lived radical intermediate species, which suggests that steric hindrance prevents the coumarin-localized radical from interacting with the backbone while simultaneously increasing the radical rearrangements with the side chains.

Mapping of Protein Disulfide Bonds Using Negative Ion Fragmentation with a Broadband Precursor Selection

Fast mapping of disulfide bonds in proteins containing multiple cysteine residues is often required in order to assess the integrity of the tertiary structure of proteins prone to degradation and misfolding or to detect distinct intermediate states generated in the course of oxidative folding. A new method of rapid detection and identification of disulfide-linked peptides in complex proteolytic mixtures utilizes the tendency of collision-activated peptide ions to lose preferentially side chains of select amino acids in the negative ion mode. Cleavages of cysteine side chains result in efficient dissociation of disulfide bonds and produce characteristic signatures in the fragment ion mass spectra. While cleavages of other side chains result in insignificant loss of mass and full retention of the peptide ion charge, dissociation of external disulfide bonds results in physical separation of two peptides and, therefore, significant changes of both mass and charge of fragment ions relative to the precursor ion. This feature allows the fragment ions generated by dissociation of external disulfide bonds to be easily detected and identified even if multiple precursor ions are activated simultaneously. Such broadband selection of precursor ions for consecutive activation is achieved by lowering the dc/rf amplitude ratio in the first quadrupole filter of a hybrid quadrupole time-of-flight mass spectrometer. The feasibility of the new method is demonstrated by partial mapping of disulfide bridges within a 37-kDa protein containing 16 cysteine residues and complete disulfide mapping within a lysozyme (14.5 kDa) containing 8 cysteine residues. In addition to detecting peptide pairs connected by a single external disulfide, the new method is also shown to be capable of identifying peptides containing both external and internal disulfide bonds. The two major factors determining the efficiency of disulfide mapping using the new methodology are the effectiveness of proteolysis and the ability of the resulting proteolytic fragments to form multiply charged negative ions.

Non‐zwitterionic structures of aliphatic‐only peptides mediated the formation and dissociation of gas phase radical cations

We have investigated how the non-zwitterionic and zwitterionic structures of aliphatic-only tripeptides affect the formation and dissociation of peptide radical cations in the gas phase. The non-zwitterionic forms of the aliphatic-only peptides in their metal complexes play an important role in determining whether the electron transfer pathway predominates. We extended this study by synthesizing permanent non-zwitterionic and zwitterionic forms of aliphatic-only peptide radical cations and exploring their reactivities in the gas phase. Collision-induced dissociation spectra demonstrated the feasibility of generating both non-zwitterionic and zwitterionic forms. Radical cations in zwitterionic forms may indeed mediate the beta and gamma carbon-carbon bond cleavages of leucine and isoleucine side chains from the GlyGlyXle radical peptides; this feature allows leucine and isoleucine residues to be distinguished unambiguously.

Backbone and Side-Chain Cleavages in Electron Detachment Dissociation (EDD)

Ab-initio electronic structure methods are used to explore potential energy profiles pertinent to the fragmentations of gas-phase radicals thought to be formed in the new negative-ion mode EDD mass spectroscopic studies of peptides. Barriers to fragmentation as well as the associated overall energy differences are computed for the observed Calpha-C backbone bond cleavage as well as for side-chain loss for a variety of side chains (valine, arginine, glutamic acid, and tyrosine). It is found that Calpha-C bond cleavage is favored over side-chain loss, although loss of a tyrosine side chain may compete with Calpha-C cleavage because the tyrosine radical formed can delocalize its unpaired electron over its aromatic ring. In addition, it is found that fragmentation of the nitrogen-centered radicals formed in EDD results in cleavage to produce so-called a*/x fragments rather than a/x* fragments both because producing the former involves a significantly smaller barrier and is nearly thermoneutral, while cleavage to yield a/x* is significantly endothermic.

Specific photodissociation of peptides with multi‐stage mass spectrometry

We report collision-induced dissociation (CID) and laser-induced dissociation (LID) performed at different wavelengths between 220 and 280 nm of the peptides leucine-enkephalin (protonated) and gramicidin A (sodiated). Hydrogen-atom losses and side-chain cleavages were observed in LID experiments. These losses depend on the laser wavelength and lead to the formation of radical ions. The fragmentations of these radicals, which are not observed in CID experiments, were investigated in multi-stage mass spectrometry experiments.

Anionic copper complex fragmentations from enkephalins under low-energy collision-induced dissociation in an ion trap mass spectrometer.

Peptide metallation with Cu2+ was explored in the negative ESI mode using an ion trap mass spectrometer. Under these conditions, the [(M-3H) + CuII]- species formed were investigated under low-energy collision-induced dissociation conditions. MS2 experiments indicate a very different behavior of CuII metallated complexes compared with [M-H]- species. CuII induces an easy loss of CO2 and specific side-chain cleavages (by radical losses) at the C-terminal residue, as observed previously by prompt 'in source' dissociation experiments. The loss of CO2 yields an unstable carbylide that leads to further dissociations involving the migration of a proton or a hydrogen radical (through the reduction of CuII). Multistage MS3 experiments were carried out to rationalize this behavior. Fragmentation pathways are proposed in order to explain the product ions observed. The side-chain radical loss at the C-terminus was demonstrated to be a consecutive process. Finally, evidence is provided that the specific side-chain cleavages can be used for the differentiation of Leu/Ile and Gln/Lys residues when they are located at the C-terminus. The existence of a zwitterionic form in the case of the anionic YGGFK-CuII complex is proposed.

Fragmentation pathways of N G-methylated and unmodified arginine residues in peptides studied by ESI-MS/MS and MALDI-MS

Protein methylation at arginine residues is a prevalent posttranslational modification in eukaryotic cells that has been implicated in processes from RNA-binding and transporting to protein sorting and transcription activation. Three main forms of methylarginine have been identified: N G-monomethylarginine (MMA), asymmetric N G,N G-dimethylarginine (aDMA), and symmetric N G,N′ G-dimethylarginine (sDMA). To investigate gas-phase fragmentations and characteristic ions arising from methylated and unmodified arginine residues in detail, we subjected peptides containing these residues to electrospray triple-quadrupole tandem mass spectrometry. A variety of low mass ions including (methylated) ammonium, carbodiimidium, and guanidinium ions were observed. Fragment ions resulting from the loss of the corresponding neutral fragments (amines, carbodiimide, and guanidine) from intact molecular ions as well as from N- and C-terminal fragment ions were also identified. Furthermore, the peptides containing either methylated or unmodified arginines gave rise to abundant fragment ions at m/ z 70, 112, and 115, for which cyclic ion structures are proposed. Electrospray ionization tandem mass spectra revealed that dimethylammonium ( m/ z 46) is a specific marker ion for aDMA. A precursor ion scanning method utilizing this fragment ion was developed, which allowed sensitive and specific detection of aDMA-containing peptides even in the presence of a five-fold excess of phosphorylase B digest. Interestingly, regular matrix-assisted laser desorption/ionization mass spectra recorded from aDMA- or sDMA-containing peptides showed metastable fragment ions resulting from cleavages of the arginine side chains. The neutral losses of mono- and dimethylamines permit the differentiation between aDMA and sDMA.

Secondary fragmentation of linear peptides in electron capture dissociation

Inspection of the electron capture dissociation (ECD) spectra of doubly-protonated peptides, Leu 4-Sar-Leu 3-Lys-OH, Leu 4-Ala-Leu 3-Lys-OH, Gly 4-Sar-Gly 3-Lys-NH 2 and Gly 3-Pro-Sar-Gly 3-Lys-NH 2, reveals extensive secondary fragmentation. In addition to w ions, entire, and in some cases multiple, cleavages of amino acid side chains from backbone fragments are observed. Extensive water loss from backbone fragments is observed for the glycine-rich peptides. For Leu 4-Ala-Leu 3-Lys, the preferred fragmentation channel is cleavage of the amide bond to produce b 7 and b 8 ions. ECD of Gly 3-Pro-Sar-Gly 3-Lys-NH 2 results in amine bond (c/z) cleavage in the proline residue accompanied by CC (or secondary NC) cleavage in the proline side chain. That fragmentation channel has not been observed previously. The peptides were also subjected to “hot” electron capture dissociation (HECD) and the resulting spectra differed markedly from those obtained under standard ECD conditions. In contrast to HECD, secondary fragmentation observed under standard ECD conditions cannot be attributed to excess energy arising from the kinetic energy of the electrons prior to capture. The results suggest that the fragmentation channels available following electron capture depend somewhat on the individual peptide structure and have mechanistic implications.

Electron capture dissociation initiates a free radical reaction cascade.

For small cyclic peptides, one electron capture by the [M + 2H](2+) ion generates numerous fragments corresponding to amino acid losses, side-chain losses, and losses of some low molecular weight species such as H(2)O, CH(3)(*), C(3)H(6), and (*)CONH(2). As predicted, the side-chain cleavages are amplified relative to linear peptides of similar size, but the amino acid losses were unexpected because they require that one electron capture cause more than one backbone cleavage, a phenomenon which necessitates further refinement or reinterpretation of current ECD mechanisms. A modified mechanism is postulated in which nonergodic electron capture fragmentation generates an alpha-carbon radical species that then propagates along the protein backbone. This radical migration initiates multiple free radical rearrangements, which cause both multiple backbone cleavages and additional side-chain cleavages.

Characterization of amino acid side chain losses in electron capture dissociation

We have used electrospray ionization (ESI) Fourier-transform ion cyclotron resonance (FTICR) mass spectrometry to characterize amino acid side chain losses observed during electron capture dissociation (ECD) of ten 7- to 14-mer peptides. Side-chain cleavages were observed for arginine, histidine, asparagine or glutamine, methionine, and lysine residues. All peptides containing an arginine, histidine, asparagine or glutamine showed the losses associated with that residue. Methionine side-chain loss was observed for doubly-protonated bombesin. Lysine side-chain loss was observed for triply-protonated dynorphin A fragment 1–13 but not for the doubly-protonated ion. The proximity of arginine to a methoxy C-terminal group significantly enhances the extent of side-chain fragmentation. Fragment ions associated with side-chain losses were comparable in abundance to those resulting from backbone cleavage in all cases. In the ECD spectrum of one peptide, the major product was due to fragmentation within an arginine side chain. Our results suggest that cleavages within side chains should be taken into account in analysis of ECD mass spectral data. Losses from arginine, histidine, and asparigine/glutamine can be used to ascertain their presence, as in the analysis of unknown peptides, particularly those with non-linear structures.

Negative ion electrospray mass spectra of caerulein peptides: an aid to structural determination.

MS/MS data derived from the [M-H](-) ions of desulfated caerulein peptides provide (i) sequencing information from a combination of alpha, beta and gamma backbone cleavages, and (ii) identification of specific amino acid side chains by side-chain cleavages [e.g. Ser (-CH(2)O), Thr (-CH(3)CHO) and Asp (-H(2)O)] (fragmentations having no counterparts in positive ion spectra). In addition, delta and/or gamma backbone cleavage ions from Asp residues identify the position of these residues in the peptide. In contrast, neither delta nor gamma cleavage ions are observed from either the Gln2 residue nor from Phe residues. Full structural information can be obtained from a consideration of the positive and negative ion MS/MS data in concert.

Negative ion fragmentations of deprotonated peptides: backbone cleavages directed through both Asp and Glu

The collision-induced spectra of [M − H]− ions of a variety of natural and synthetic amphibian peptides containing Asp and/or Glu exhibit characteristic γ backbone cleavage ions that identify the positions of these residues in the peptide. A theoretical study suggests that the Glu cleavage involves an SNi reaction of the carboxylate anion from the Glu α side chain to form a deprotonated cyclic lactone. The presence of either Asp or Glu or other residues that effect pronounced side-chain cleavages (e.g. Ser or Thr) results in the normal α and β backbone cleavages being reduced in comparison to those cleavages which originate from side chains. Copyright © 2001 John Wiley & Sons, Ltd.

Electrospray/collision-induced dissociation of derivatised peptides: studies on a hybrid magnetic sector–orthogonal time-of-flight mass spectrometer

A series of peptides C-terminally derivatised with 4-aminonaphthalenesulphonic acid has been analysed on a hybrid magnetic sector–orthogonal time-of-flight mass spectrometer by negative-ion electrospray with collision-induced dissociation. The fragmentation reactions of 400 eV (laboratory frame of reference) [M–H] − projectile ions in collisions with helium, argon, xenon and methane target gases have been investigated. With all the collision gases, C-terminal fragment ions predominate; side-chain cleavages were observed with xenon as the collision gas, but not with helium. The collision gas can be rapidly changed from helium to xenon, thereby giving spectra containing complementary sequence information from picomole amounts of sample.

Effects of heavy gases on the tandem mass spectra of peptide ions in the quadrupole ion trap

Heavy gases (xenon, argon, krypton, methane) have been used to improve the performance of the quadrupole ion trap when performing collision-induced dissociation on peptides. MS/MS spectra reveal that increased amounts of internal energy can be deposited into peptide ions and more structural information can be obtained. Specifically, the pulsed introduction of the heavy gases (as reported previously by Doroshenko, V. M.; Cotter, R. J. Anal. Chem. 1996, 68, 463) provides greater energy deposition without the deleterious effects that static pressures of heavy gas have on spectra. Internal energy deposition as indicated by a qualitative evaluation of MS/MS spectra shows pulsed introduction of heavy gases enables ions to obtain more internal energy than possible by using static pressures of the same heavy gases. A linear correlation is observed between the percentage of heavy gas added and the ratio of product ions used to reflect internal energy deposition. Results here also show that upon pulsed introduction of heavy gases, empirical optimization of a single frequency resonant excitation signal is no longer needed to obtain good MS/MS spectrometry efficiency. The presence of many low mass-to-charge ratio ions and the absence of side chain cleavages in the MS/MS spectra of peptides suggests that the propensity for consecutive fragmentations is increased with the pulsed introduction of heavy gases. In addition, by varying the delay time between introduction of the gas and application of the resonant excitation signal, the amount of fragmentation observed in MS/MS spectra can be changed.

Fragmentation of protonated peptides: surface-induced dissociation in conjunction with a quantum mechanical approach

This paper describes the results of a systematic investigation designed to assess the utility of surface-induced dissociation in the structural analysis of small peptides (500-1800u). A number of different peptides, ranging in mass and amino acid sequence, are fragmented by collision with a surface in a tandem mass spectrometer and the spectra are compared with data obtained by gas-phase collisional activation. The surface-induced dissociation spectra provide ample sequence information for the peptides. Side-chain cleavage ions of type w, which are generally detected upon kiloelectronvolt collisions with gaseous targets but not upon electronvolt collisions with gaseous targets, are detected in the ion-surface collision experiments. A theoretical approach based on MNDO bond order calculations is suggested for the description of peptide fragmentation. This model, supplemented by ab initio calculations, serves as a complement to the experimental work described in the paper and explains (i) the easy cleavage of the amide bond, (ii) charge-remote backbone and side-chain cleavages, and (iii) the influence of intramolecular H-bonding.

An expanded nomenclature scheme for labeling peptide fragmentations and its use with 'AMASS', a computer program for generating all possible fragment ion structures from known precursors.

A nomenclature scheme for exhaustively labeling peptide fragment ions is proposed. The scheme is based on IUPAC nomenclature and the previously proposed Roepstorff nomenclature scheme used to label fragment ions in linear peptides. The descriptor used is specifically defined in order to increase the number of peptide and side chain linkages to which the nomenclature scheme can be applied compared with the Roepstorff scheme. The proposed descriptor can be used unambiguously to assign all possible fragments from linear, cyclized, branched, extended (i.e. beta-amino acids) and retro inverso peptides. A significant advantage of the proposed scheme is its simple interface with the currently accepted Roepstorff scheme. This nomenclature scheme is able to label all theoretical fragments generated by the computer program 'AMASS'. AMASS is proposed as a means of systematically calculating the mass of all possible fragment ions from known precursor structures. The program can help determine whether a peptide fragment was derived from an internal sequence fragment or a combination of side chain and backbone cleavages. The program AMASS and the proposed nomenclature scheme are used to illustrate a procedure for identifying fragment ions in the metastable product ion spectrum of somatostatin-14. We envisage that this procedure will be useful for identifying fragment ions which are characteristic of particular structural arrangements in dicyclic and polycyclic peptides.

A biomarker study of petroleum from the Neogene Tertiary sedimentary basins in Northeast Japan.

Crude oils and condensates from the Japanese Neogene Tertiary sedimentary basins were analyzed for steroid and triterpenoid hydrocarbons, using GC/MS. Most crude oils show incomplete side chain epimerization of C29 steranes. This is possibly due to an insufficient reaction time and temperature for epimerization since the Miocene age. All oils show a good correlation between nuclear isomerization and side chain epimerization of C29 steranes. The correlation curve appears to be different from that previously reported, which may have resulted from a difference in thermal histories. On the basis of the extents of side chain cleavages of saturated and triaromatic steroid hydrocarbons as well as upon the extents of configurational isomerizations of steranes and hopanes, the condensates are considered to be distinctly maturer than the crude oils. This fact suggests that the condensates have undergone higher temperatures than the crude oils. Among the crude oils, those from the Niigata basin are significantly maturer than those from the Akita-Yamagata basin. This is probably due to the difference between the oil source rocks in the two basins, which may cause a difference in the stage of primary migration of oils. The conversion of monoaromatic to triaromatic steroid hydrocarbons does not correlate well with the side chain epimerization of steranes. This may be due to preferred migration of monoaromatics by geochromatographic separation. There is a significant difference between the crude oils from the Niigata basin and those from the Akita-Yamagata basin in respect of the C27/C29 ratios of steroid hydrocarbons. This difference is consistent with the previous report on visual kerogen compositions of oil source rocks from these basins. The relative abundance of C27 to C29 of monoaromatic steroid hydrocarbons is thought to be less affected by thermal stress than that of steranes. This could thus be a more useful organic source indicator in the case off matured oils such as condensates in Japanese Tertiary sedimentary basins.

A comparison of electron impact and field ionization spectra of some 2,5‐diketopiperazines

AbstractThe mass spectra of cyclo‐(Gly‐Leu), cyclo‐(Gly‐Tyr), cyclo‐(Ala‐Tyr), cyclo‐(Leu‐Tyr), cyclo‐(Val‐Phe) and cyclo‐(Leu‐Phe) are presented. The use of high resolution mass measurements and metastable analysis allows the proposal of some general fragmentation mechanisms. The dominant features in the mass spectra of 2,5‐diketopiperazines are fragments corresponding to cleavages of sidechains and the rupture of the piperazine‐2,5‐dione ring. The characteristic fragments may be used in the identification of any 2,5‐diketopiperazines by mass spectrometry.

Acth and sulfatase activity

The conversion of 5-en-3β-ol steroid sulfates to active hormones has been extensively studied. A preliminary desulfation of the precursors is required to allow their structure 5-ene-3β (-ol to be transformed to the configuration 4-ene-3-keto present in active hormones. Steroid sulfatase is a very active enzyme but present at a very low concentration with respect to other steroid biosynthesizing enzymes in the adrenal tissue. It represents a limiting enzyme reaction in corticoid production from early precursors. Steroid sulfatase hydrolyzes pregnenolone-sulfate (P-S) much more effectively than DHEA-sulfate (D-S). Its activity and concentration in the adrenal is increased by in vivo stimulation with ACTH, however, ACTH does not seem to stimulate other enzyme reactions after free pregnenolone. Both P-S and D-S act on the steroid sulfatase inhibiting reciprocally the desulfation of the other, in such a way that when both sulfates are present simultaneously, P-S is efficiently hydrolyzed to release free P which is quickly converted to progesterone and corticoids, while most of D-S remains as sulfate and is excreted as such. Measurements of the endogeneous pools of steroid precursors in the adrenal show that, ACTH stimulation decreases the levels of P-S, while free P increases first and then decreases as it is converted to progesterone and corticoids. ACTH may have two parallel but distinct mechanisms of action on corticoid biosynthesis. One, already established, is through the activation of adenylcyclase, c-AMP release and a final increment of TPNH to favor adrenal hydroxylations and side chain cleavages. The other, to be demonstrated as yet, suggests a stimulation of steroid sulfatase without c-AMP participation and regulates the amount of free 5-en-3β-ol steroid available for active hormone biosynthesis.