Virtual screening and rational design of antioxidant peptides based on tryptophyllin L structures isolated from the Litoria rubella frog.

Discovery of natural antioxidants has been carried out for decades relying mainly on experimental approaches that are commonly associated with time and cost demanding biochemical assays. The maturation of quantitative structure activity relationship (QSAR) modelling has provided an alternative approach for searching and designing antioxidant compounds with alleviated costs. As a contribution to this approach, this work aimed to establish a fragment-based 3D-QSAR procedure to discover and design potential antioxidants based on tryptophyllin L structures isolated from the red tree frog Litoria rubella. A force field and a Gaussian 3D-QSAR model were built to screen for potential antioxidants from tripeptide fragments covering all sequences of tryptophyllin L database. Among those, PWY(NH2 ) corresponding tryptophyllin L 4.1 was predicted to have the highest 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) radical cation (ABTS+ ·) scavenging capability. Two newly designed peptides PYW and PYW(NH2 ) together with PWY(NH2 ), tryptophyllin L 4.1, and the reference peptide PWY were synthesized and subjected to two antioxidant assays including ABTS scavenging and ferric reducing antioxidant power assays. Although the experimental TEAC values of the five peptides were roughly similar to those from predictions, the activity order was not in agreement with the predictions. The dissimilarities were accounted by the difference in the experimental procedures, the deviation of modelling regression, and the synergetic effect of structural and experimental features. The ABTS radical scavenging assays revealed that all the tested peptides were strong ABTS+ · scavengers with the antioxidant capabilities approximately twice as high as trolox and higher than glutathione. The ferric reducing activities of the peptides were, on the other hand, much weaker than that of trolox suggesting different antioxidant mechanisms inserted by trolox and the peptides. This work was a demonstration that 3D-QSAR methods can be employed in conjunction with experimental methods to effectively detect and design antioxidant peptides.

Negative ion cleavages of (M-H)- anions of peptides. Part 3. Post-translational modifications.

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.

The Amyloid Fibril-Forming Properties of the Amphibian Antimicrobial Peptide Uperin†3.5.

The amphibian skin is a vast resource for bioactive peptides, which form the basis of the animals' innate immune system. Key components of the secretions of the cutaneous glands are antimicrobial peptides (AMPs), which exert their cytotoxic effects often as a result of membrane disruption. It is becoming increasingly evident that there is a link between the mechanism of action of AMPs and amyloidogenic peptides and proteins. In this work, we demonstrate that the broad-spectrum amphibian AMP uperin 3.5, which has a random-coil structure in solution but adopts an α-helical structure in membrane-like environments, forms amyloid fibrils rapidly in solution at neutral pH. These fibrils are cytotoxic to model neuronal cells in a similar fashion to those formed by the proteins implicated in neurodegenerative diseases. The addition of small quantities of 2,2,2-trifluoroethanol accelerates fibril formation by uperin 3.5, and is correlated with a structural stabilisation induced by this co-solvent. Uperin 3.5 fibril formation and the associated cellular toxicity are inhibited by the polyphenol (-)-epigallocatechin-3-gallate (EGCG). Furthermore, EGCG rapidly dissociates fully formed uperin 3.5 fibrils. Ion mobility-mass spectrometry reveals that uperin 3.5 adopts various oligomeric states in solution. Combined, these observations imply that the mechanism of membrane permeability by uperin 3.5 is related to its fibril-forming properties.

Structural analysis of calmodulin binding by nNOS inhibitory amphibian peptides.

Calmodulin (CaM) is a ubiquitous protein in nature and plays a regulatory role in numerous biological processes, including the upregulation of nitric oxide (NO) synthesis in vivo. Several peptides that prevent NO production by interacting with CaM have been isolated in the cutaneous secretions of Australian amphibians, and are thought to serve as a defense mechanism against predators. In this work, we probe the mechanism by which three of these peptides, namely, caerin 1.8, dahlein 5.6, and a synthetic modification of citropin 1.1, interact with CaM to inhibit NO signaling. Isothermal titration calorimetry was used to determine thermodynamic parameters of the binding interactions and revealed that all the peptides bind to CaM in a similar fashion, with the peptide encapsulated between the two lobes of CaM. Ion mobility-mass spectrometry was used to investigate the changes in collision cross section that occur as a result of complexation, providing additional evidence for this binding mode. Finally, nuclear magnetic resonance spectroscopy was used to track chemical shift changes upon binding. The results obtained confirm that these complexes adopt canonical collapsed structures and demonstrate the strength of the interaction between the peptides and CaM. An understanding of these molecular recognition events provides insights into the underlying mechanism of the amphibian host-defense system.

The identification of disulfides in ricin D using proteolytic cleavage followed by negative-ion nano-electrospray ionization mass spectrometry of the peptide fragments.

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.

Fragmentations of [M-H]- anions of peptides containing Ser sulfate. A joint experimental and theoretical study.

RATIONALE: To determine the negative-ion cleavages from [M-H](-) ions of Ser sulfate-containing peptides using experiment and theory in concert. METHODS: Fragmentations were explored using a Waters QTOF2 mass spectrometer in negative-ion electrospray mode, together with calculations at the CAM-B3LYP/6-311++g(d,p) level of theory. Peptides used in this study were: GS(SO3H)(OH) 1 GS(SO3H)(OCH3) 1a GAVS(SO3H)(OH) 2 GAVS(SO3H)(OCH3) 2a GLS(SO3H)(GVA(OH) 3 GLS(SO3H)GDA(OH) 4 GLS(SO3H)GS(SO3H)A(OH) 5. RESULTS: Previously, it has been shown that a peptide containing a Tyr sulfate group shows [(M-H)(-) -SO3] as the base peak. Only a small peak was observed corresponding to HOSO3(-) (formed following rearrangement of the sulfate). A Ser sulfate-containing peptide, in contrast, shows pronounced peaks due to cleavage product anions [(M-H)(-)-SO3] and HOSO3(-). Theoretical calculations at the CAM-B3LYP/6-311++g(d,p) level of theory suggest that rearrangement of a Ser sulfate to give C-terminal CO2SO3H is energetically unfavourable in comparison with fragmentation of the intact Ser sulfate to yield [(M-H)(-)-SO3] and HOSO3(-). [(M-H)(-)-H2SO4] anions are not observed in the spectra of peptides containing Ser sulfate, presumably because HOSO3(-) is a relatively weak gas-phase base (ΔGacid = 1265 kJ mol(-1)). CONCLUSIONS: Experimental and theoretical data suggest that [(M-H)(-)-SO3] and HOSO3(-) product anions (from a peptide with a C-terminal Ser sulfate) are formed from the serine sulfate anion accompanied by specific proton transfer. CID MS/MS/MS data for an [(M-H)(-)-SO3] ion of an underivatised sulfate-containing peptide will normally allow the determination of the amino acid sequence of that peptide. The one case we have studied where that is not the case is GLS(SO3H)GDA(OH), where the peptide contains Ser sulfate and Asp, where the diagnostic Asp cleavages are competitive with the Ser sulfate cleavages.

Negative ion fragmentations of disulfide-containing cross-linking reagents are competitive with aspartic acid side-chain-induced cleavages.

RATIONALE: It has been shown that the disulfide moiety in the chemical cross-linking reagent dithiobis(succinimidyl)propionate (DSP), which is similar in structure to the natural cystine disulfide, cleaves preferentially to the peptide backbone in the negative ion mode. However, the tandem mass (MS/MS) spectra of peptides in the negative ion mode are often dominated by products arising from low-energy, side-chain-induced processes, which may compete with any facile cross-linker fragmentations and complicate identification of chemical cross-links in a complex mixture. METHODS: Two disulfide-containing crosslinking reagents similar to DSP, but with varying spacer arm lengths, were synthesized and the MS/MS spectra of several model peptides cross-linked with these reagents were investigated. Theoretical calculations were used to describe the energetics of the cross-linker fragmentations as well as several low-energy side-chain-induced fragmentations which compete with disulfide cleavages. RESULTS: Altering the spacer arm length of the cross-linker, such that there is one methylene group less than in DSP, results in a more facile cleavage process, whilst the opposite is true when a methylene group is added. Of the low-energy side-chain-induced fragmentations studied, only those from aspartic acid compete significantly with those of the cross-linker disulfide. CONCLUSIONS: Low-energy cleavage processes from aspartic acid that compete with cross-linker fragmentations occur in the negative ion MS/MS spectra of the cross-linked peptides, irrespective of the spacer arm length. Other fragmentation pathways do not significantly interfere with low-energy disulfide cleavage, making the presence of additional product ions in the MS/MS spectrum diagnostic for the presence of aspartic acid.

Fragmentations of [M-H]- anions of peptides containing tyrosine sulfate. Does the sulfate group rearrange? A joint experimental and theoretical study.

RATIONALE: To investigate the fragmentations in the negative-ion electrospray mass spectra of peptides containing tyrosine sulfate. METHODS: Possible fragmentation mechanisms were explored using a Waters QTOF2 tandem mass spectrometer in concert with calculations at the CAM-B3LYP/6-311++g(d,p) level of theory. RESULTS: The major negative ion formed in the ESI-MS of peptides containing tyrosine sulfate is [(M-H)-SO3](-) and this process normally yields the base peak of the spectrum. The basic backbone cleavages of [(M-H)-SO3](-) allowed the sequence of the peptide to be determined. Rearrangement reactions involving the formation of HOSO3(-) and [(M-H)-H2SO4](-) yielded minor peaks with relative abundances ≤ 10% and ≤ 2%, respectively. CONCLUSIONS: The mass spectra of the [M-H](-) and [(M-H)-SO3](-) anions of peptides containing tyrosine sulfate allowed the position of the tyrosine sulfate group to be determined, together with the amino acid sequence of the peptide.

Pheromones, attractants and other chemical cues of aquatic organisms and amphibians.

This review covers the subject of pheromones, attractants and other chemical cues of aquatic invertebrates, fishes and amphibians (including salamanders and anurans). Major topics include the sex pheromones of gastropods, salamanders and a giant tree frog, and the conspecific attraction of sperm to ova of some of the organisms and animals described in this review.

Studies of cyclization reactions of linear cumulenes and heterocumulenes using the neutralization-reionization procedure and/or ab initio calculations.

A number of linear cumulenes and heterocumulenes have been made by charge stripping of anions of known bond connectivity in the source of a mass spectrometer. Some of these reactive molecules have been identified in interstellar molecular clouds. The structures of these neutrals may be investigated by reionization to a decomposing positive ion [the neutralization-reionization technique ((-)NR(+))], and/or by ab initio calculations. Energized linear cumulenes and heterocumulenes may undergo cyclization to form stable cyclic isomers. To cite a selection of the examples described in this review: (i) four-atom systems CCCC and some heterocumulenes CCCX (X=B, N, Al, Si, P) involve the formation of stable four-membered ring rhombic (also called kite and fan) structures. One of the cyclic molecules, cyclo-C(3) Si, has been detected in interstellar molecular clouds, (ii) five-atom cumulene and heterocumulene systems are more complex. Linear CCCCC rearranges the carbon skeleton by forming a C substituted rhomboid system, CCCCO forms a three-membered cyclic isomer, while nitrogen containing five-atom cumulenes effect nitrile to isonitrile interconversion via three-centered cyclized intermediates, and (iii) CCCCCC and CCCCBO cyclize to give unique six-membered ring systems.

Backbone fragmentations of [M-H]- anions from peptides. Reinvestigation of the mechanism of the beta prime cleavage.

RATIONALE: An experimental study has shown that the structure of a ß' ion proposed earlier is incorrect. Backbone cleavage ß' anions have structures R(NH(-)) from systems [[RNHCH(X)CONHCH(Y)CO(2)H (or C-terminal CONH(2))-H](-) (where R is the rest of the peptide molecule and X and Y represent the α side chains of the individual amino acid residues). METHODS: Ab initio calculations were carried out at the CAM-B3LYP/6-311++g(d,p) level of theory. CONCLUSIONS: The calculations suggest that RNH(-) ions are formed by S(N)i cyclisation processes involving either (i) the C-terminal CO(2)(-) or C-terminal [CONH](-) as appropriate, or (ii) an enolate ion [-NHC(-)(Y)-] cyclising at the backbone CH of the -CH(X)- group. Concomitant C-N bond cleavage then liberates an RNH(-) ion, processes which can occur along the peptide backbone.

Hydrogen tunnelling influences the isomerisation of some small radicals of interstellar importance. A theoretical investigation.

Hydrogen atom isomerisations within five radical systems (i.e., CH(3)˙NH/˙CH(2)NH; CH(3)O˙/˙CH(2)OH; ˙CH(2)SH/CH(3)S˙; CH(3)CO(2)˙/˙CH(2)CO(2)H; and HOCH(2)CH(2)O˙/HO˙CHCH(2)OH) have been studied via quantum-mechanical hydrogen tunnelling through reaction barriers. The reaction rates including hydrogen tunnelling effects have been calculated for these gas phase reactions at temperatures from 300 K to 0 K using Wenzel-Kramers-Brillouin (WKB) and Eckart methods. The Eckart method has been found to be unsatisfactory for the last two systems listed above, because it significantly underestimates the width of the reaction barriers for the interconversions. The calculations at all-electron CCSD(T)/CBS level of theory indicate that the barriers for all reactions (forward and reverse) are greater than 100 kJ mol(-1), meaning that the chemical reactivity of the reactants is limited in the absence of hydrogen tunnelling. Hydrogen tunnelling, in some cases, enhance rates of reaction by more than 100 orders of magnitude at low temperature, and around 2 orders of magnitude at room temperature, compared to results obtained from canonical variational transition state theory. Tunnelling corrected reaction rates suggest that some of these isomerisation reactions may occur in interstellar media.

Can cytosine, thymine and uracil be formed in interstellar regions? A theoretical study.

This theoretical study investigates possible synthetic routes to cytosine, uracil and thymine in the gas phase from precursor molecules that have been detected in interstellar media. Studies at the CCSD(T)/6-311++G(d,p)//B3LYP/6-311++G(d,p) level of theory suggest that: The reactions between :CCCNH and :CCCO with monosolvated urea may constitute viable interstellar syntheses of cytosine and uracil. No low energy equilibration between cytosine and uracil has been demonstrated. The interaction of :CH(2) with the 5 C-H bond of uracil may form thymine in an energetically favourable reaction, but competing reactions where :CH(2) reacts with double bonds and other CH and NH bonds of uracil, reduce the effectiveness of this synthesis. The reaction between the hydrated propional enolate anion and isocyanic acid may produce thymine, in a reaction sequence where ΔG(reaction)(298 K) is -22 kJ mol(-1) and the maximum energy requirement (barrier to the first transition state) is only 47 kJ mol(-1).

QCM-D fingerprinting of membrane-active peptides.

The increasing prevalence of antibiotic-resistant bacteria is becoming a public health crisis. Antimicrobial peptides (AMPs) are a promising solution, because bacterial resistance is less likely. Quartz crystal microbalance with dissipation monitoring (QCM-D) is a versatile and valuable technique for investigation of these peptides. This article looks at the different approaches to the interpretation of QCM-D data, showing how to extract the maximum information from the data. Five AMPs of diverse charge, length and activity are used as case studies: caerin 1.1 wild-type, two caerin 1.1 mutants (Gly15Gly19-caerin 1.1 and Ala15Ala19-caerin 1.1), aurein 1.2 and oncocin. The interaction between the AMP and a 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) membrane is analysed inter alia using frequency-dissipation plots (∆f-∆D plots) to ascertain the mechanism of action of the AMP. The ∆f-∆D plot can then be used to provide a fingerprint for the AMP-membrane interaction. Building up a database of these fingerprints for all known AMPs will enable the relationship between AMP structure and membrane activity to be better understood, hopefully leading to the future development of antibiotics without bacterial resistance.

Diagnostic cyclisation reactions which follow phosphate transfer to carboxylate anion centres for energised [M-H]- anions of pTyr-containing peptides.

The low-energy negative ion phosphoTyr to C-terminal -CO(2)PO(3)H(2) rearrangement occurs for energised peptide [M-H](-) anions even when there are seven amino acid residues between the pTyr and C-terminal amino acid residues. The rearranged C-terminal -CO(2)PO(2)H(O(-)) group effects characteristic S(N)i cyclisation/cleavage reactions. The most pronounced of these involves the electrophilic central backbone carbon of the penultimate amino acid residue. This reaction is aided by the intermediacy of an H-bonded intermediate in which the nucleophilic and electrophilic reaction centres are held in proximity in order for the S(N)i cyclisation/cleavage to proceed. The ΔG(reaction) is +184 kJ mol(-1) with the barrier to the S(N)i transition state being +240 kJ mol(-1) at the HF/6-31 + G(d)//AM1 level of theory. A similar phosphate rearrangement from pTyr to side chain CO(2)(-) (of Asp or Glu) may also occur for energised peptide [M-H](-) anions. The reaction is favourable: ΔG(reaction) is -44 kJ mol(-1) with a maximum barrier of +21 kJ mol(-1) (to the initial transition state) when Asp and Tyr are adjacent. The rearranged species R(1)-Tyr-NHCH(CH(2)CO(2)PO(3)H(-))COR(2) (R(1) = CHO; R(2) = OCH(3)) may undergo an S(N)i six-centred cyclisation/cleavage reaction to form the product anion R(1)-Tyr(NH(-)). This process has a high energy requirement [ΔG(reaction) = +224 kJ mol(-1), with the barrier to the S(N)i transition state being +299 kJ mol(-1)].

Can collision-induced negative-ion fragmentations of [M-H](-) anions be used to identify phosphorylation sites in peptides?

A joint experimental and theoretical investigation of the fragmentation behaviour of energised [M-H](-) anions from selected phosphorylated peptides has confirmed some of the most complex rearrangement processes yet to be reported for peptide negative ions. In particular: pSer and pThr (like pTyr) may transfer phosphate groups to C-terminal carboxyl anions and to the carboxyl anion side chains of Asp and Glu, and characteristic nucleophilic/cleavage reactions accompany or follow these rearrangements. pTyr may transfer phosphate to the side chains of Ser and Thr. The reverse reaction, namely transfer of a phosphate group from pSer or pThr to Tyr, is energetically unfavourable in comparison. pSer can transfer phosphate to a non-phosphorylated Ser. The non-rearranged [M-H](-) species yields more abundant product anions than its rearranged counterpart. If a peptide containing any or all of Ser, Thr and Tyr is not completely phosphorylated, negative-ion cleavages can determine the number of phosphated residues, and normally the positions of Ser, Thr and Tyr, but not which specific residues are phosphorylated. This is in accord with comments made earlier by Lehmann and coworkers.

Histidine-containing host-defence skin peptides of anurans bind Cu2+. An electrospray ionisation mass spectrometry and computational modelling study.

Anuran peptides which contain His, including caerin 1.8 (GLFKVLGSVAKHLLPHVVPVIAEKL-NH(2)), caerin 1.2 (GLLGVLGSVAKHVLPHVVPVIAEHL-NH(2)), Ala(15) maculatin 1.1 (GLFGVLAKVAAHVVAIEHF-NH(2)), fallaxidin 4.1 (GLLSFLPKVIGHLIHPPS-OH), riparin 5.1 (IVSYPDDAGEHAHKMG-NH(2)) and signiferin 2.1 (IIGHLIKTALGMLGL-NH(2)), all form MMet(2+) and (M + Met(2+)-2H(+))(2+) cluster ions (where Met is Cu, Mg and Zn) following electrospray ionisation (ESI) in a Waters QTOF 2 mass spectrometer. Peaks due to Cu(II) complexes are always the most abundant relative to other metal complexes. Information concerning metal(2+) connectivity in a complex has been obtained (at least in part) using b and y fragmentation data from ESI collision-induced dissociation tandem mass spectrometry (CID MS/MS). Theoretical calculations, using AMBER version 10, show that MCu(2+) complexes with the membrane active caerin 1.8, Ala(15) maculatin 1.1 and fallaxidin 4.1 are four-coordinate and approximating square planar, with ligands including His and Lys, together with the carbonyl oxygens of particular backbone amide groups. When binding can occur through two His, or one His and one Lys, the His/Lys ligand structure is the more stable for the studied systems. The three-dimensional (3D) structures of the complexes are always different from the previously determined structures of the uncomplexed model peptides (using 2D nuclear magnetic resonance (NMR) spectroscopy in membrane-mimicking solvents like trifluoroethanol/water).

Diagnostic di- and triphosphate cyclisation in the negative ion electrospray mass spectra of phosphoSer peptides.

It has been shown previously that [M-H](-) anions of small peptides containing two phosphate residues undergo cyclisation of the phosphate groups, following collision-induced dissociation (CID), to form a characteristic singly charged anion A (H3P2O7(-), m/z 177). In the present study it is shown that the precursor anions derived from the diphosphopeptides of caerin 1.1 [GLLSVLGSVAKHVLPHVVPVIAEHL(NH2)] and frenatin 3 [GLMSVLGHAVGNVLGGLFKPKS(OH)] also form the characteristic product anion A (m/z 177). Both of the precursor peptides show random structures in water, but partial helices in membrane-mimicking solvents [e.g. in d3-trifluoroethanol/water (1:1)]. In both cases the diphosphopeptide precursor anions must have flexible conformations in order to allow approach of the phosphate groups with consequent formation of A: for example, the two pSer groups of 4,22-diphosphofrenatin 3 are seventeen residues apart. Finally, CID tandem mass spectrometric (MS/MS) data from the [M-H](-) anion of the model triphosphoSer-containing peptide GpSGLGpSGLGpSGL(OH) show the presence of both product anions A (m/z 177) and D (m/z 257, H4P3O10(-)). Ab initio calculations at the HF/6-31+G(d)//AM1 level of theory suggest that cyclisation of the three phosphate groups occurs by a stepwise cascade mechanism in an energetically favourable reaction (ΔG = -245 kJ mol(-1)) with a maximum barrier of +123 kJ mol(-1).

An unusual kynurenine-containing opioid tetrapeptide from the skin gland secretion of the Australian red tree frog Litoria rubella. Sequence determination by electrospray mass spectrometry.

The Kyn-containing peptide FP-Kyn-L(NH(2)) is an unusual minor component of the skin peptide profile of the Australian red tree frog Litoria rubella collected from an area within a 20 kilometre radius of Alice Springs in central Australia. The structure was determined by electrospray mass spectrometry and synthesis. The major component of the skin secretion is the analogous tryptophyllin peptide FPWL(NH(2)). Both peptides show opioid activity at 10(-7) M, and are likely to act via the µ opioid receptor.

Fragmentations of (M-H)- anions of underivatised peptides. Part 2: Characteristic cleavages of Ser and Cys and of disulfides and other post-translational modifications, together with some unusual internal processes.

In a previous review (Bowie, Brinkworth, & Dua (2002); Mass Spectrom Rev 21:87-107) we described the characteristic backbone cleavages and side chain fragmentations which occur from (M-H)(-) parent anions of underivatized peptides. This work is briefly summarized in the present review. Cys was not described in the previous review: here we describe the Cys characteristic side chain loss of H(2)S, together with its gamma backbone cleavage. These processes are compared with those of the related Ser. All experimental observations are backed up with theoretical studies at the HF/6-31G(d)//AM1 level of theory, a level of theory which we have shown gives good geometries and acceptable relative energies. The negative ion cleavages of a number of post-translational modifications are described. Negative ion mass spectrometry is the method of choice for identification of disulfides in both peptides and proteins. Intramolecular disulfides are identified by the presence of the fragment anion [(M-H)(-)-H(2)S(2)], and CID MS2 of this fragment normally identifies the positions of the two Cys residues and often the full sequence of the peptide. An unsymmetrically substituted intermolecular disulfide can give up to eight characteristic fragment anions, and CID MS2 of some, or all of these often provides the full sequence of those peptides which form the initial intermolecular disulfide linkage. Negative ion cleavages of disulfides are the most energetically favored of all peptide negative cleavages studied to date. Negative ion mass spectrometry is also valuable for the identification of pyroglutamates, sulfates and phosphates. Finally, some unusual fragmentations are described which involve cyclization/elimination reactions which require the decomposing (M-H)(-) parent anions to adopt the same helical conformation that these peptides have in solution.