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It is now 25 years since we commenced the study of the negative-ion fragmentations of peptides and we have recently concluded this research with investigations of the negative-ion chemistry of most post-translational functional groups. Our first negative-ion peptide review (Bowie, Brinkworth, & Dua, 2002) dealt with the characteristic backbone fragmentations and side-chain cleavages from (M-H) - ions of underivatized peptides, while the second (Bilusich & Bowie, 2009) included negative-ion backbone cleavages for Ser and Cys and some initial data on some post-translational groups including disulfides. This third and final review provides a brief summary of the major backbone and side chain cleavages outlined before (Bowie, Brinkworth, & Dua, 2002) and describes the quantum mechanical hydrogen tunneling associated with some proton transfers in enolate anion/enolate systems. The review then describes, in more depth, the negative-ion cleavages of the post-translational groups Kyn, isoAsp, pyroglu, disulfides, phosphates, and sulfates. Particular emphasis is devoted to disulfides (both intra- and intermolecular) and phosphates because of the extensive and spectacular anion chemistry shown by these groups. © 2016 Wiley Periodicals, Inc. Mass Spec Rev., (© 2016 Wiley Periodicals, Inc.)

Rationale: To use negative-ion nano-electrospray ionization mass spectrometry of peptides from the tryptic digest of ricin D, to provide sequence information; in particular, to identify disulfide position and connectivity., Methods: Negative-ion fragmentations of peptides from the tryptic digest of ricin D was studied using a Waters QTOF2 mass spectrometer operating in MS and MS(2) modes., Results: Twenty-three peptides were obtained following high-performance liquid chromatography and studied by negative-ion mass spectrometry covering 73% of the amino-acid residues of ricin D. Five disulfide-containing peptides were identified, three intermolecular and two intramolecular disulfide-containing peptides. The [M-H](-) anions of the intermolecular disulfides undergo facile cleavage of the disulfide units to produce fragment peptides. In negative-ion collision-induced dissociation (CID) these source-formed anions undergo backbone cleavages, which provide sequencing information. The two intramolecular disulfides were converted proteolytically into intermolecular disulfides, which were identified as outlined above., Conclusions: The positions of the five disulfide groups in ricin D may be determined by characteristic negative-ion cleavage of the disulfide groups, while sequence information may be determined using the standard negative-ion backbone cleavages of the resulting cleaved peptides. Negative-ion mass spectrometry can also be used to provide partial sequencing information for other peptides (i.e. those not containing Cys) using the standard negative-ion backbone cleavages of these peptides., (Copyright © 2014 John Wiley & Sons, Ltd.)

A method using headspace solid-phase microextraction (HS-SPME) followed by gas chromatography/mass spectrometry (GC/MS) analysis has been developed to gain insight into the degradation of the chemical warfare agent sulfur mustard in solution. Specifically, the described approach simplifies the sample preparation for GC/MS analysis to provide a rapid determination of changes in sulfur mustard abundance. These results were found to be consistent with those obtained using liquid-liquid extraction (LLE) GC/MS. The utility of the described approach was further demonstrated by the investigation of the degradation process in a complex matrix with surfactant added to assist solvation of sulfur mustard. A more rapid reduction in sulfur mustard abundance was observed using the HS-SPME approach with surfactant present and was similar to results from LLE experiments. Significantly, this study demonstrates that HS-SPME can simplify the sample preparation for GC/MS analysis to monitor changes in sulfur mustard abundance in solution more rapidly, and with less solvent and reagent usage than LLE., (Copyright © 2010 Commonwealth of Australia.)

Ricin is a toxic protein produced in the seeds of the castor bean plant. The toxicity of the protein and the ease in which it can be extracted from the seeds makes it a potential biological warfare agent. There has been extensive work in the development of analytical techniques that can identify the protein robustly and rapidly. On-target tryptic digestion and MALDI MS was used to distinguish ricin from bovine serum albumin and three other type 2 ribsome-inactivating proteins (RIPs), abrin, agglutinin (RCA(120)), and viscumin, using the peptide mass fingerprint. The sequence coverage obtained was enhanced using methanol-assisted tryptic digestion and was particularly useful for the detection of these toxins in complex matrixes. When used in conjunction with intact protein MALDI mass measurement, a positive identification of ricin (or any of the other RIPs) was achieved including confirmation of the integrity of the disulfide bond between the A and B chains. This applicability of this methodology was demonstrated by the identification of ricin in a typical "crude white powder" that may be illicitly produced in a clandestine lab. The analysis on the solubilized sample using this method can be undertaken in around an hour with minimal sample preparation.

Ricin is a highly toxic protein from the seeds of the castor bean plant. Crude extracts from castor beans are toxic by several routes, and there is international concern about the use of these extracts by terrorist organizations. Lethality in aerosolized form has spurred the development of methods for the rapid detection of this protein from air samples that is critical in determining the illicit use of this material. Matrix-assisted laser desorption ionization (MALDI) mass measurement with an automated laser firing sequence was used to detect intact ricin from solutions containing less than 4 microg/mL of ricin in the presence of other endogenous seed proteins. This sensitivity was attained with the addition of 0.01% Tween 80 to the extracts that greatly enhanced the ricin signal. Importantly, this treatment substantially reduces the interference from the castor bean seed storage proteins. Commonly the ricin signal can be completely obscured by the oligomers of seed storage proteins, and this treatment reveals the ricin molecular ion, allowing the analyst to make a judgment as to the ricin content of the extract. This method provides for sensitive and rapid identification of intact ricin from aqueous samples with little sample preparation and is amenable to automatic acquisition.

The ubiquitous nature of protein phosphorylation makes it challenging to map kinase-substrate relationships, which is a necessary step toward defining signaling network architecture. To trace the activity of individual kinases, we developed a semisynthetic reaction scheme, which results in the affinity tagging of substrates of the kinase in question. First, a kinase, engineered to use a bio-orthogonal ATPgammaS analog, catalyzes thiophosphorylation of its direct substrates. Second, alkylation of thiophosphorylated serine, threonine or tyrosine residues creates an epitope for thiophosphate ester-specific antibodies. We demonstrated the generality of semisynthetic epitope construction with 13 diverse kinases: JNK1, p38alpha MAPK, Erk1, Erk2, Akt1, PKCdelta, PKCepsilon, Cdk1/cyclinB, CK1, Cdc5, GSK3beta, Src and Abl. Application of this approach, in cells isolated from a mouse that expressed endogenous levels of an analog-specific (AS) kinase (Erk2), allowed purification of a direct Erk2 substrate.

Buffers and detergents are notorious for suppression of analyte signal in electrospray and MALDI mass spectrometry and, invariably, analysts will take steps to remove these contaminants before MS analysis. However, we have found serendipitously that protein signal with MALDI MS is improved by about an order of magnitude on the addition of small amounts of Tween80. Four charged states of BSA could easily be seen at less than 125 fmol/spot and with mixture of three proteins (BSA, trypsinogen, and protein A) the molecular ions could be detected on as little as 12.5 fmol of spotted material (per protein) using an automated laser firing sequence.

Recently, we showed that the signal intensity of intact protein by matrix-assisted laser desorption/ionisation (MALDI) mass spectro-metry measurement can be enhanced at least an order of magnitude by the addition of Tween80 to the analyte solution. We did not ascertain whether this effect was limited to Tween80 or if it was more universal of biological detergents. This paper discusses our investigations into this question. A variety of chemically diverse detergents were added to analyte solutions containing bovine serum albumin (BSA) to determine whether there was significant signal enhancement. The addition of Tween20, Tween80, Triton X100 and Triton X-114 improved the attainable sensitivity of intact protein MALDI mass spectrometry compared to spectra acquired without detergent. In some cases there was considerable improvement in signal--for example, with Triton X-100 two charge states (the +1 and +2) of BSA (3.9 fmol) could easily be observed. Another advantage of this process is that the detergent can be added directly to the matrix solution reducing sample handling and preparation time. We propose this phenomenon results from the ability of these detergents to increase the solubility of the protein via hydrophobic and hydrophilic interactions between the detergent and protein. The increased solubility allows for more uniform deposition of the analyte/-matrix mixtures producing an evenly distributed layer of analyte especially useful for data acquisition using an automated laser firing sequence.

The recent development of novel fragmentation processes based on either electron capture directly or transfer from an anion show great potential for solving problems in proteomics that are intractable by the more widely employed thermal-based fragmentation processes such as collision induced dissociation. The dominant fragmentation occurring upon electron capture dissociation of peptides is cleavage of N-C alpha bonds in the peptide backbone to form c and z* ions. In the case of disulfide-linked peptides, it has also been shown that electron capture on one of the cystine sulfur atoms is favored, resulting in cleavage of the disulfide bond. In this study, we report that electron capture on the sulfur of alkylated cysteine residues is also a dominant process, causing cysteine side-chain loss from z* ions.

The skin secretion of the Dainty Green Tree Frog Litoria gracilenta contains 16 peptides, which protect the animal from predators, both large and small. A combination of negative and positive ion electrospray mass spectrometry together with Lys-C enzymic digest and Edman sequencing identifies three new wide-spectrum caerin 1 antibiotics, namely Caerin 1.17 [GLFSVLGSVAKHLLPHVAPIIAEKL-NH2], Caerin 1.18 [GLFSVLGSVAKHLLPHVVPVIAEKL-NH2], and Caerin 1.19 [GLFKVLGSVAKHLLPHVAPIIAEKL-NH2], and a narrow spectrum antibiotic Caerin 3.5 [GLWEKVKEKANELVSGIVEGVK-NH2].

Eugenin [pGluGlnAspTyr(SO(3))ValPheMetHisProPhe-NH(2)] has been isolated from the pouches of female Tammar wallabies (Macropus eugenii) carrying young in the early lactation period. The sequence of eugenin has been determined using a combination of positive and negative ion electrospray mass spectrometry. This compound bears some structural resemblance to the mammalian neuropeptide cholecystokinin 8 [AspTyr(SO(3))MetGlyTrpMetAspPhe-NH(2)] and to the amphibian caerulein peptides [caerulein: pGluGlnAspTyr(SO(3))ThrGlyTrpMetAspPhe-NH(2)]. Eugenin has been synthesized by a route which causes only minor hydrolysis of the sulfate group when the peptide is removed from the resin support. Biological activity tests with eugenin indicate that it contracts smooth muscle at a concentration of 10(-9) M, and enhances the proliferation of splenocytes at 10(-7) M, probably via activation of CCK(2) receptors. The activity of eugenin on splenocytes suggests that it is an immunomodulator peptide which plays a role in the protection of pouch young.

[M--H]- parent anions of underivatised peptides containing an intramolecular disulfide bridge undergo characteristic loss of the elements of H2S2, a process diagnostic of the presence of the disulfide moeity. This facile process is initiated from a side-chain enolate anion. Theoretical calculations (at the HF/6-31G(d)//AM1 level of theory) indicate that the process is exothermic with a small barrier. When the disulfide link involves a C-terminal Cys, the negative ion spectrum shows an [(M--H)--(H2S2+CO2)] fragment anion which is usually the main peak of the spectrum. This process is also directed by an enolate anion: theoretical calculations suggest a stepwise sequence with loss of CO2 preceding loss of H2S2. Both [(M--H)--H2S2] and [(M--H)--(H2S2+CO2)] anions undergo backbone cleavage allowing identification of the amino acid sequence of the peptide., ((c) 2005 John Wiley & Sons, Ltd.)

The secretion from the dorsal glands of the frog Litoria rothii contains a series of new peptides including rothein 1 (SVSNIPESIGF-OH, a neuropeptide which contracts smooth muscle), a number of inactive rothein 2 and 3 peptides (e.g. rothein 2.1, AGGLDDLLEPVLNSADNLVHGL-OH), and a new proline rich peptide, named rothein 4.1 (AEILFGDVRPPWMPPPIFPEMP-OH), which shows neither antimicrobial nor neuronal nitric oxide synthase (nNOS) activity. Two known neuropeptides of the caerulein family [e.g. caerulein, pEQDY(SO3)TGWMDF-NH2] together with a series of known caerin 1 antibiotic and nNOS-inhibiting peptides (e.g. caerin 1.1, GLLSVLGSVAKHVLPHVVPVIAEHL-NH2) were also identified. Positive ion electrospray mass spectrometry (ES-MS) was used as the primary method to investigate the sequences of the new peptides. Negative ion ES-MS was used to fill in any gaps in the positive ion data and, finally, Edman automated sequencing was used to differentiate between Leu and Ile and to confirm the sequences determined by mass spectrometry., (Copyright (c) 2005 John Wiley & Sons, Ltd.)

Host-defence peptides secreted from the skin glands of Australian frogs and toads, are, with a few notable exceptions, different from those produced by anurans elsewhere. This review summarizes the current knowledge of the following classes of peptide isolated and characterized from Australian anurans: neuropeptides (including smooth muscle active peptides, and peptides that inhibit the production of nitric oxide from neuronal nitric oxide synthase), antimicrobial and anticancer active peptides, antifungal peptides and antimalarial peptides. Other topics covered include sex pheromones of anurans, and the application of peptide profiling to (i). recognize particular populations of anurans of the same species and to differentiate between species, and (ii). investigate evolutionary aspects of peptide formation.

Caerin 1.1 is a potent broad-spectrum antibacterial peptide isolated from a number of Australian frogs of the Litoria genus. In membrane-like media, this peptide adopts two alpha-helices, separated by a flexible hinge region bounded by Pro15 and Pro19. Previous studies have suggested that the hinge region is important for effective orientation of the two helices within the bacterial cell membrane, resulting in lysis via the carpet mechanism. To evaluate the importance of the two Pro residues, they were replaced with either Ala or Gly. The antibacterial activity of these two peptides was tested, and their three-dimensional structures were determined using two-dimensional NMR spectroscopy and restrained molecular dynamics calculations. The resulting structures indicate that the central hinge angle decreases significantly upon replacement of the Pro residues with Gly and to a further extent with Ala. This trend was mirrored by a corresponding decrease in antibiotic activity, further exemplifying the necessity of the hinge in caerin 1.1 and related peptides. In a broader context, the use of Pro, Gly, and Ala variants of caerin 1.1 has enabled the relationship between conformational flexibility and activity to be directly investigated in a systematic manner.

The Cys residue initiates characteristic backbone cleavages of [M-H](-) anions of Cys-containing peptides. A combination of experiment and theory suggests that these processes are initiated by molecular recognition between the C-terminal CONH(-) group (in this study all peptides have C-terminal CONH(2) groups) and the SH in the Cys side chain to form an S-H...O=C hydrogen bond. This process is exothermic by 60 kJ mol(-1) (calculations at the HF/6-31G(d)//AM1 level of theory). The structure of this reactive intermediate has the NH(-) of the amide group and the central CH of the Cys residue locked into position such that these groups effect an S(N)2 process to form an intermediate which can either (i) dissociate to give an RNH(-) species [the delta ion (process endothermic by 37 kJ mol(-1) with a barrier of 132 kJ mol(-1))], or (ii) effect deprotonation within the intermediate to eliminate RNH(2) to give the gamma backbone cleavage anion in a reaction exothermic by 40 kJ mol(-1) with a barrier of 132 kJ mol(-1). Collision-induced mass spectra of the [M-H](-) anions of five selected Cys-containing peptides all contain gamma and (gamma-H(2)S) anions. Three of these spectra also show the less favoured delta cleavage anions., (Copyright 2004 John Wiley & Sons, Ltd.)

The amino acid sequences of seven signiferin peptides are provided by consideration of tandem mass spectrometric (MS/MS) data for the respective MH+ and [M--H]- precursor ions. These methods do not differentiate between isomeric residues Leu and Ile; these were identified using the Edman degradation technique. The sequence of signiferin 1, a new smooth muscle contracting neuropeptide (RLCIPYIIPC-OH) containing a disulfide bridge, is best determined using the collision-induced dissociation (CID) spectrum of the [M--H]- anion. The initial fragmentation of this system involves loss of H2S2, which furnishes an open-chain system that is readily sequenced using the alpha and beta backbone cleavage anions., (Copyright 2004 John Wiley & Sons, Ltd.)

The peptide frenatin 3 is a major component of the skin secretion of the Australian giant tree frog, Litoria infrafrenata. Frenatin 3 is 22 amino acids in length, and shows neither antimicrobial nor anticancer activity. It inhibits the production of nitric oxide by the enzyme neuronal nitric oxide synthase at a micromolar concentration by binding to its regulatory protein, Ca2+ calmodulin, a protein known to recognize and bind amphipathic alpha-helices. The solution structure of frenatin 3 has been investigated using NMR spectroscopy and restrained molecular dynamics calculations. In trifluoroethanol/water mixtures, the peptide forms an amphipathic alpha-helix over residues 1-14 while the C-terminal eight residues are more flexible and less structured. The flexible region may be responsible for the lack of antimicrobial activity. In water, frenatin 3 exhibits some alpha-helical character in its N-terminal region., (Copyright 2003 Wiley Periodicals, Inc.)

A large number of bioactive peptides have been isolated from amphibian skin secretions. These peptides have a variety of actions including antibiotic and anticancer activities and the inhibition of neuronal nitric oxide synthase. We have investigated the structure-activity relationship of citropin 1.1, a broad-spectrum antibiotic and anticancer agent that also causes inhibition of neuronal nitric oxide synthase, by making a number of synthetically modified analogues. Citropin 1.1 has been shown previously to form an amphipathic alpha-helix in aqueous trifluoroethanol. The results of the structure-activity studies indicate the terminal residues are important for bacterial activity and increasing the overall positive charge, while maintaining an amphipathic distribution of residues, increases activity against Gram-negative organisms. Anticancer activity generally mirrors antibiotic activity suggesting a common mechanism of action. The N-terminal residues are important for inhibition of neuronal nitric oxide synthase, as is an overall positive charge greater than three. The structure of one of the more active synthetic modifications (A4K14-citropin 1.1) was determined in aqueous trifluoroethanol, showing that this peptide also forms an amphipathic alpha-helix.

Collision-induced fragmentations of deprotonated maculatin 1 peptides provide significant sequencing information. When the peptide lacks those residues which can fragment through their alpha side chains (e.g. Thr, Ser, Glu and Gln in this study) the basic alpha and beta' backbone cleavages occur from the [Mbond;H](-) anion. When Thr, Ser, Glu and Gln are present, the ease of side-chain fragmentation of these residues is: Thr (loss of MeCHO) > Ser (CH(2)O) > Glu (H(2)O) > Gln (NH(3)). When one of more of these residues is (are) present, the alpha and beta' cleavages often occur from a fragment rather than the [Mbond;H](-) anion, e.g. for Thr, the [(Mbond;H)(-)bond;MeCHO](-) anion. These four residues also initiate gamma backbone cleavage reactions. The relative abundances of peaks resulting from gamma cleavage are Glu > Ser = Thr > Gln for maculatin 1 spectra. An unusual Gln19/Ile17 cyclisation/cleavage reaction occurs in maculatin spectra: the peptide [Mbond;H](-) anion must adopt a helical conformation in order for these two groups to interact. Analogous fragmentations have been reported previously in the negative ion spectra of the caerin 1 peptides., (Copyright 2003 John Wiley & Sons, Ltd.)

Loss of H2S is the characteristic Cys side-chain fragmentation of the [M-H]- anions of Cys-containing peptides. A combination of experiment and theory suggests that this reaction is initiated from the Cys enolate anion as follows: RNH-(-)C(CH2SH)CONHR' Ø [RNHC(=CH2)CONHR' (HS-)] Ø [RNHC(=CH2)CO-HNR'-H]-+H2S. This process is facile. Calculations at the HF/6-31G(d)//AM1 level of theory indicate that the initial anion needs only > or =20.1 kcal mol(-1) of excess energy to effect loss of H2S. Loss of CH2S is a minor process, RNHCH(CH2SH)CON(-)-R' Ø RNHCH(CH2S-)CONHR' Ø RNH -CHCONHR+CH2S, requiring an excess energy of > or =50.2 kcal mol(-1). When Cys occupies the C-terminal end of a peptide, the major fragmentation from the [M-H]- species involves loss of (H2S+CO2). A deuterium-labelling study suggests that this could either be a charge-remote reaction (a process which occurs remote from and uninfluenced by the charged centre in the molecule), or an anionic reaction initiated from the C-terminal CO2- group. These processes have barriers requiring the starting material to have an excess energy of > or =79.6 (charge-remote) or > or =67.1 (anion-directed) kcal mol(-1), respectively, at the HF/6-31G(d)//AM1 level of theory. The corresponding losses of CH2O and H2O from the [M-H]- anions of Ser-containing peptides require > or =35.6 and > or =44.4 kcal mol(-1) of excess energy (calculated at the AM1 level of theory), explaining why loss of CH2O is the characteristic side-chain loss of Ser in the negative ion mode., (Copyright 2003 John Wiley & Sons, Ltd.)

This article describes the fundamental cleavage reactions of (M-H)(-) anions of underivatized peptides that contain up to 25 amino acid residues. The experimental observations of these cleavages have been backed up by molecular modeling, generally at the AM1 level of theory. The basic cleavages are the ubiquitous alpha- and beta-backbone cleavage reactions, which provide information similar to that of the B and Y + 2 cleavages of MH(+) ions of peptides. The residues Asp and Asn also effect cleavages of the backbone (called delta- and gamma-cleavages), by reactions initiated from side chain enolate anions, causing elimination reactions that cleave the backbone between the Asp (Asn) N bond;C backbone bond. Glu and Gln also direct analogous delta- and gamma-cleavages of the backbone, but in this case the processes are initiated by attack of the side chain CO(2) (-) (CONH(-)) to form a lactone (lactam). Ser and Thr residues undergo characteristic fragmentations of the side chain. These processes, losses of CH(2)O (Ser) and MeCHO (Thr), convert these residues into Gly. In larger peptides, Ser and Thr can effect two backbone cleavage reactions, called gamma- and epsilon -processes. The C-terminal CO(2) (-) (or CONH(-)) forms a hydrogen bond with the side chain OH (of Ser or Thr), placing the C-terminal residue in a position where it may affect S(N) (2) attack at the electrophilic backbone CH of Ser, with concomitant cleavage of the backbone. All of the above negative ion cleavages require the peptide backbone to be conformationally flexible. However, there is a backbone cleavage that requires the peptide to have an alpha-helical conformation in order for the two reacting centers to approach. This cleavage is illustrated for the Glu 23-initiated backbone cleavage at Ile 21 for the (M-H)(-) anion of the antimicrobial peptide caerin 1.1., (Copyright 2002 Wiley Periodicals, Inc.)

This paper reports the study of backbone cleavages in the collision-induced negative-ion mass spectra of the [M - H](-) anions of some synthetic modifications of the bioactive amphibian peptide citropin 1 (GLFDVIKKVASVIGGL-NH(2)). The peptides chosen for study contain no amino acid residues which could effect facile side-chain cleavage, i.e. Ser (-CH(2)O, side-chain cleavage) and Asp (-H(2)O) are replaced by Ala or Lys. We expected that such peptides should exhibit standard and pronounced peaks due to alpha cleavage ions (and to a lesser extent beta cleavage ions) in their collision-induced negative-ion spectra. This expectation was realised, but the spectra also contained peaks formed by a new series of cleavage anions. These are produced following cyclisation of the C-terminal CONH(-) moiety at carbonyl functions of amide groups along the peptide backbone; effectively transferring the NH of the C-terminal CONH(-) group to other amino acid residues. We have called the product anions of these processes beta' ions, in order to distinguish them from standard beta ions. Some beta' ions also fragment directly to some other beta' ions of smaller mass. The reaction coordinates of alpha,beta and beta' backbone processes have been calculated at the HF/6-31G*//AM1 level theory for simple model systems. The initial cyclisation step of the beta' sequence is barrierless and exothermic. Subsequent steps have a maximum barrier of +40 kcal mol(-1), with the overall reaction being endothermic by some 30 kcal mol(-1) at the level of theory used. These calculations take no account of the complexity of the conformationally flexible peptide system, and it is surprising that each of the two reacting centres can 'find' each other in such a large system., (Copyright 2002 John Wiley & Sons, Ltd.)

The positive ion electrospray mass spectra of [M+H](+) and the negative ion electrospray mass spectra of [M-H](-) ions of selected pyroglutamate containing peptides both provide sequencing data. The negative ion spectra show the normal alpha and beta backbone cleavages in addition to delta and gamma backbone cleavages initiated by the side chains of Glu and Phe residues. For example, the [M-H](-) ion of pGlu Pro Gln Val Phe Val-NH(2) shows delta and gamma peaks at m/z 224 (delta, Gln3), 244 (gamma, Phe4), 451 (delta, Phe4), 471 (gamma, Gln3). Some of the negative ion spectra show unusual grandaughter peaks that originate by alpha and beta, or delta and gamma backbone cleavages of a beta(1) cleavage ion., (Copyright 2002 John Wiley & Sons, Ltd.)

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., (Copyright 2002 John Wiley & Sons, Ltd.)

The collision induced spectra of [M - H](-) anions from of caerin 1 peptides and some synthetic modifications show the usual alpha, beta and beta' backbone cleavages together with Ser (epsilon,gamma) and Glu (gamma) cleavages which break the peptide backbone in the vicinity of those residues. All of these cleavages require the peptide backbone to be flexible. There is also a backbone cleavage of a type not observed before. This cleavage involves nucleophilic attack of the carboxylate anion of the Glu23 side chain at the backbone CH of Ile 21. We propose that this cleavage requires the caerin peptide to be in an alpha helical conformation (the 3D structure that this peptide adopts in solution) in order that the interacting groups are held in close proximity., (Copyright 2002 John Wiley & Sons, Ltd.)

Two neuropeptides have been isolated and identified from the secretions of the skin glands of the Stony Creek Frog Litoria lesueuri. The first of these, the known neuropeptide caerulein 1.1, is a common constituent of anuran skin secretions, and has the sequence pEQY(SO3)TGWMDF-NH2. This neuropeptide is smooth muscle active, an analgaesic more potent than morphine and is also thought to be a hormone. The second neuropeptide, a new peptide, has been named lesueurin and has the primary structure GLLDILKKVGKVA-NH2. Lesueurin shows no significant antibiotic or anticancer activity, but inhibits the formation of the ubiquitous chemical messenger nitric oxide from neuronal nitric oxide synthase (nNOS) at IC(50) (16.2 microm), and is the first amphibian peptide reported to show inhibition of nNOS. As a consequence of this activity, we have tested other peptides previously isolated from Australian amphibians for nNOS inhibition. There are three groups of peptides that inhibit nNOS (IC(50) at microm concentrations): these are (a) the citropin/aurein type peptides (of which lesueurin is a member), e.g. citropin 1.1 (GLFDVIKKVASVIGGL-NH(2)) (8.2 microm); (b) the frenatin type peptides, e.g. frenatin 3 (GLMSVLGHAVGNVLG GLFKPK-OH) (6.8 microm); and (c) the caerin 1 peptides, e.g. caerin 1.8 (GLFGVLGSIAKHLLPHVVPVIAEKL-NH(2)) (1.7 microm). From Lineweaver-Burk plots, the mechanism of inhibition is revealed as noncompetitive with respect to the nNOS substrate arginine. When the nNOS inhibition tests with the three peptides outlined above were carried out in the presence of increasing concentrations of Ca(2+) calmodulin, the inhibition dropped by approximately 50% in each case. In addition, these peptides also inhibit the activity of calcineurin, another enzyme that requires the presence of the regulatory protein Ca(2+) calmodulin. It is proposed that the amphibian peptides inhibit nNOS by interacting with Ca(2+)calmodulin, and as a consequence, blocks the attachment of this protein to the calmodulin domain of nNOS.

Eleven dahlein peptides are present in the skin secretion of the Australian aquatic frog Litoria dahlii. All peptides have been sequenced using a combination of electrospray mass spectrometry (ES-MS) and Lys-C digestion/MS, with each sequence confirmed by automated Edman sequencing. The 13-residue dahlein 1 peptides (e.g. dahlein 1.1 GLFDIIKNIVSTL-NH(2)) exhibit weak wide-spectrum antimicrobial activity but no significant activity in the anticancer testing program of the National Cancer Institute (Washington). There are no potent antimicrobial peptides present in the glandular secretion, but the dahleins 5 strongly inhibit the formation of NO by neuronal nitric oxide synthase (e.g. dahlein 5.1 GLLGSIGNAIGAFIANKLKP-OH)., (Copyright 2001 John Wiley & Sons, Ltd.)

The collision-induced spectra of [M - H](-) ions of a variety of natural and synthetic amphibian peptides containing Asp and/or Glu exhibit characteristic gamma backbone cleavage ions that identify the positions of these residues in the peptide. A theoretical study suggests that the Glu cleavage involves an S(N)i reaction of the carboxylate anion from the Glu alpha 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 alpha and beta backbone cleavages being reduced in comparison to those cleavages which originate from side chains., (Copyright 2001 John Wiley & Sons, Ltd.)