Albumin Oxidation and Albumin Glycation Discordance During Type 2 Diabetes Therapy: Biological and Clinical Implications.

Aims: Cys34 albumin redox modifications (reversible "cysteinylation" and irreversible "di/trioxidation"), besides being just oxidative stress biomarkers, may have primary pathogenetic roles to initiate and/or aggravate cell, tissue, and vascular damage in diabetes. In an exploratory "proof-of-concept" pilot study, we examined longitudinal changes in albumin oxidation during diabetes therapy. Methods: Mass spectrometric analysis was utilized to monitor changes in human serum albumin (HSA) post-translational modifications {glycation [glycated albumin (GA)], cysteinylation [cysteinylated albumin (CA) or human non-mercaptalbumin-1; reversible], di/trioxidation (di/trioxidized albumin or human non-mercaptalbumin-2; irreversible), and truncation (truncated albumin)} during ongoing therapy. Four informative groups of subjects were evaluated [type 1 diabetes (T1DM), type 2 diabetes (T2DM), prediabetes-obesity, and healthy controls] at baseline, and subjects with diabetes were followed for a period up to 280 days. Results: At baseline, T2DM was associated with relatively enhanced albumin cysteinylation (CA% total) compared with T1DM (P = 0.004), despite comparable mean hyperglycemia (P values: hemoglobin A1c = 0.09; GA = 0.09). T2DM, compared with T1DM, exhibited selectively and significantly higher elevations of all the "individual" glycated cum cysteinylated ("multimodified") albumin isoforms (P values: CysHSA+1G = 0.003; CysHSA+2G = 0.007; and CysHSA+3G = 0.001). Improvements in glycemic control and decreases in albumin glycation during diabetes therapy in T2DM were not always associated with concurrent reductions of albumin cysteinylation, and in some therapeutic situations, albumin cysteinylation worsened (glycation-cysteinylation discordance). Important differences were observed between the effects of sulfonylureas and metformin on albumin molecular modifications. Conclusions: T2DM was associated with higher oxidative (cysteinylation) and combined (cysteinylation plus glycation) albumin molecular modifications, which are not ameliorated by improved glucose control alone. Further studies are required to establish the clinical significance and optimal therapeutic strategies to address oxidative protein damage and resulting consequences in diabetes.

Cysteine-free cone snail venom peptides: Classification of precursor proteins and identification of mature peptides.

The cysteine-free acyclic peptides present in marine cone snail venom have been much less investigated than their disulfide bonded counterparts. Precursor protein sequences derived from transcriptomic data, together with mass spectrometric fragmentation patterns for peptides present in venom duct tissue extracts, permit the identification of mature peptides. Twelve distinct gene superfamiles have been identified with precursor lengths between 64 and 158 residues. In the case of Conus monile, three distinct mature peptides have been identified, arising from two distinct protein precursors. Mature acyclic peptides are often post-translationally modified, with C-terminus amidation, a feature characteristic of neuropeptides. In the present study, 20 acyclic peptides from Conus monile and Conus betulinus were identified. The common modifications of C-terminus amidation, gamma carboxylation of glutamic acid (E to ϒ), N-terminus conversion of Gln (Q) to a pyroglutamyl residue (Z), and hydroxylation of Pro (P) to Hyp (O) are observed in one or more peptides identified in this study. Proteolytic trimming of sequences by cleavage at the C-terminus of Asn (N) residues is established. The presence of an asparagine endopeptidase is strengthened by the identification of legumain-like sequences in the transcriptome assemblies from diverse Conus species. Such sequences may be expected to have a cleavage specificity at Asn-Xxx peptide bonds.

Differential Spectrum of Albumin Glycation, Oxidation, and Truncation in Type 2 and Type 1 Diabetes: Clinical and Biological Implications.

Background: Albumin, the most abundant and physiologically vital serum protein, accumulates a range of chemical modifications, as consequence of encounters with large number of reactive molecules whose concentrations increase in serum under pathological conditions. Methods: In a "proof of concept" study, mass spectrometric analysis was utilized to quantitate albumin post-translational modifications (glycation, oxidation, and truncation; individual isoforms and total) in four informative subject groups [type 1 diabetes (T1DM), type 2 diabetes (T2DM), prediabetes-obesity and healthy; all with estimated glomerular filtration rate ≥60 mL/(min·m2)]. Besides glycated albumin (GA/mass spectrometry), glycated serum protein (GSP/nitro blue tetrazolium colorimetry), and glycated hemoglobin (HbA1c/high-performance liquid chromatography) were also measured. Results: A wide spectrum of albumin molecular modifications was identified in diabetes, with significant differences between T2DM and T1DM. Albumin glycation: GA correlated more strongly with HbA1c in T1DM, compared to T2DM. Higher albumin glycation isoforms (human serum albumin +3G/2G) were more stable and discriminative markers of mean glycemia. Albumin oxidation: T2DM, in comparison with T1DM, showed enhanced oxidative and dual (glycation plus oxidation) modifications, representing extreme molecular pathology. Albumin truncation: There was dramatic reduction ("deficiency") of truncated albumin isoforms in T2DM, and significant reduction in T1DM. Albumin truncation negatively correlated with severity of albumin glycation (mean glycemia) and albumin oxidation (cysteinylation). Possible mechanisms of insulin resistance, with associated increased free fatty acids binding to albumin, in stimulating albumin oxidation and inhibiting albumin glycation ("metabolic cross talks") are reviewed. Conclusions: Albumin molecular modifications in diabetes, together with significant differences between T2DM and T1DM, suggest possible role for insulin resistance in their genesis and consequent cell, tissue, and vascular dysfunction/damage. Albumin molecular fingerprinting can provide valuable insights into pathogenesis, diagnosis, monitoring, and future therapies for diabetes. Identification of biomarker battery ("albuminomics," "diabetomics") driven diverse "healthy," prediabetes, obesity, and T2DM phenotypes represents additional novel step toward precision medicine in diabetes and related disorders.

Mechanistic Investigations on N-Cα Bond Cleavages in Dibasic Peptides Containing Internal Lys and Arg Residues.

The model nonapeptide AAARAAKAG* (* indicates amide) is used to explore N-Cα bond fragmentation under CID-MS conditions. Neighboring group participation and the effect of positioning of Lys and Arg residues on N-Cα bond cleavage is established using a library of synthetic peptide analogues. The importance of the Arg residue at position 4 and the i to i+3 spacing between Arg and Lys residues in determining the formation of the N-Cα bond cleaved product ions (cn) is demonstrated by a comparative MS study of positional variants in analogue peptides. The effect of shortening of the Lys side chain has been established using ornithine (Orn) and diaminobutyric acid (Dab) analogues. The involvement of the Lys residue in mediating the N-Cα bond cleavage is further probed using Nε-dimethyl and isotopically labeled 15Nα, Îµ lysine residues. MSn experiments reveal that the c6 ion originates from a doubly charged dehydrated b8 ion [b8-18]2+. The mechanism of this unusual fragmentation process has been probed by using position 8 analogues (Gly, Ala, and Aib). A plausible mechanism is proposed for the origin of the c6 ion, which involves C-terminus lactam formation followed by transannular cyclization and dehydration. The results presented in this study highlight the role of reactive side chain functionalities in promoting noncanonical fragmentation pathways.

Mechanistic Insights into an Unusual Side-Chain-Mediated N-Cα Bond Cleavage under Collision-Induced Dissociation Conditions in the Disulfide-Containing Peptide Conopressin.

Conopressin, a nonapeptide disulfide CFIRNCPKG amide present in cone snail venom, undergoes a facile cleavage at the Cys6-Pro7 peptide bond to yield a disulfide bridged b6 ion. Analysis of the mass spectral fragmentation pattern reveals the presence of a major fragment ion, which is unambiguously assigned as the tripeptide sequence IRN amide. The sequence dependence of this unusual fragmentation process has been investigated by comparing it with the fragmentation patterns of related peptides, oxytocin (CYIQNCPLG amide), Lys-vasopressin (CYFQNCPKG amide), and a series of synthetic analogues. The results establish the role of the Arg4 residue in facilitating the unusual N-Cα bond cleavage at Cys6. Structures are proposed for a modified disulfide bridged fragment containing the Cys1 and Cys6 residues. Gas-phase molecular dynamics simulations provide evidence for the occurrence of conformational states that permit close approach of the Arg4 side chain to the Cys6 Cß methylene protons.

Pregabalin peptides: conformational comparison of γ3- and γ4-substituted γ-amino acids in αγααα pentapeptides.

Gamma-aminobutyric acid (GABA, gammaAbu), an unsubstituted gamma-amino acid, is an important inhibitory neurotransmitter in the mammalian brain. The role of GABA in the treatment of epilepsy has triggered a great deal of interest in substituted gamma-amino acids, which may serve as GABA analogs, acting as inhibitors of GABA aminotransferase. Pregabalin (Pgn), a well-known antiepileptic drug, is also a beta-substituted gamma3-amino acid. Pregabalin and gamma4Leu, an isomer of the pregabalin (Pgn) residue, both carrying the same isobutyryl group in the side chain, were introduced in the present study to have a comparison of their respective conformational differences as well as their role in influencing the overall conformation of the peptides, they are inserted in. Two alpha-gamma-alpha-alpha-alpha hybrid pentapeptides were designed that contain Aib-Pgn and Aib-gamma4Leu segments at the N terminus. The study provides a detailed analysis of the conformational properties and non-covalent interactions observed in the crystal structures of two polymorphs of the pentapeptide monohydrate, Boc-Aib-(S)Pgn-Leu-Phe-Val-OMe (C38H63N5O8·H2O) and the isomeric pentapeptide, Boc-Aib-gamma4(R)Leu-Leu-Phe-Val-OMe (C38H63N5O8), obtained from single crystal X-ray diffraction experiments.

Cone Snail Glutaminyl Cyclase Sequences from Transcriptomic Analysis and Mass Spectrometric Characterization of Two Pyroglutamyl Conotoxins.

The post-translational modification of N-terminal glutamine (Q) to a pyroglutamyl (Z) residue is observed in the conotoxins produced by marine cone snails. This conversion requires the action of the enzyme glutaminyl cyclase (QC). Four complete QC sequences from the species C. araneosus, C. frigidus, C. litteratus, and C. monile and two partial sequences from C. amadis and C. miles have been obtained by analysis of transcriptomic data. Comparisons with mammalian enzyme sequences establish a high level of identity and complete conservation of functional active site residues, including a cluster of hydrogen-bonded acidic side chains. Mass spectrometric analysis of crude venom samples coupled to conotoxin precursor protein sequences obtained from transcriptomic data establishes the presence of pyroglutamyl conotoxins in the venom of C. frigidus and C. amadis. The C. frigidus peptide belongs to the M superfamily, with cysteine framework III, whereas the C. amadis peptide belongs to the divergent superfamily with cysteine framework VI/VII. Additionally, gamma carboxylation of glutamic acid and hydroxylation of proline are observed in the C. frigidus peptide. Mass spectral data are available via ProteomeXchange with identifier PXD009006.

Contryphan Genes and Mature Peptides in the Venom of Nine Cone Snail Species by Transcriptomic and Mass Spectrometric Analysis.

The occurrence of contryphans, a class of single-disulfide-bond-containing peptides, is demonstrated by the analysis of the venom of nine species of cone snails. Ten full gene sequences and two partial gene sequences coding for contryphan precursor proteins have been identified by next-generation sequencing and compared with available sequences. The occurrence of mature peptides in isolated venom has been demonstrated by LC-ESI-MS/MS analysis. De novo sequencing of reduced, alkylated contryphans from C. frigidus and C. araneosus provides evidence of sequence variation and post-translational modification, notably gamma carboxylation of glutamic acid. The characterization of Fr965 (C. frigidus) provides a rare example of a sequence lacking Pro at position 5 in the disulfide loop. The widespread occurrence of contryphan genes and mature peptides in the venom of diverse cone snails is suggestive of their potential biological significance.

Intramolecular backbone···backbone hydrogen bonds in polypeptide conformations. The other way around: ɛ-turn.

In this study, we performed a detailed literature survey of the ɛ-turn in peptides and proteins. This three-dimensional structural feature is characterized by an eleven-membered pseudo-cycle closed by an intramolecular backbone…backbone H-bond. Interestingly, in this motif the direction of the N-H…O = C H-bond runs opposite to that of the much more popular and extensively investigated α-, ß-, and γ-turns. We did not authenticate unequivocally the ɛ-turn main-chain reversal topology in any linear short peptide. However, it is frequently observed in small cyclic peptides formed by four, five, and six amino acid residues with stringent geometric requirements. Rather surprisingly, ɛ-turns do occur in proteins, although to a relatively moderate extent, as an isolated feature or in the turn segment of hairpin motifs based on two antiparallel, pleated ß-strands. Moreover, the ɛ-turn may also host not only the seven-membered, intramolecularly H-bonded, pseudo-cycle termed γ-turn, either of the classic or inverse type, but also one (or even two) cis peptide bond(s) or a ß-bulge conformation. Based on their ϕ, ψ backbone torsion angles, we were able to classify the protein ɛ-turns in six different families. Conformational energy computations using the DFT methodology were also performed on the ɛ-turns adopted by the amino acid triplet -Gly-Gly-Gly- (Gly is the most commonly found residue at each of the three positions in our analysis of proteins). Again, in this computational study, six families of turns were identified, but only some of them resemble rather closely those extracted from our investigation on proteins.

Homooligomeric ß3 (R)-valine peptides: Transformation between C14 and C12 helical structures induced by a guest Aib residue.

Novel helical, structures unprecedented in the chemistry of α-polypeptides, may be found in polypeptides containing ß and γ amino acids. The structural characterization of C12 and C14 -helices in oligo ß-peptides was originally achieved using conformationally constrained cyclic ß-residues. This study explores the conformational characteristics of proteinogenic ß3 residues in homooligomeric sequences and addresses the issue of inducing a transition between C14 and C12 helices by the introduction of a guest α-residue. Folded C14 -helical structures are demonstrated for the nonapeptide Boc-[ß3 (R)Val]9 -OMe by NMR methods in CDCl3 -DMSO mixtures, while the peptide was found to be aggregated in CDCl3 . The insertion of a guest Aib residue into an oligo-ß-valine sequence in the octapeptide model Boc-[(ß3 (R)Val)3 -Aib-(ß3 (R)Val]4 -OMe results in well dispersed NH region in the NMR spectrum indicating folded structures in CDCl3 . Structure calculations for both the peptides using NOE distance constraints support a C14 helical structure in the homooligomer which transform into a C12 helix on introduction of the guest Aib residue.

Crystallographic characterization of the α,γ C12 helix in hybrid peptide sequences.

The solid-state conformations of two αγ hybrid peptides Boc-[Aib-γ(4) (R)Ile]4 -OMe 1 and Boc-[Aib-γ(4) (R)Ile]5 -OMe 2 are described. Peptides 1 and 2 adopt C12 -helical conformations in crystals. The structure of octapeptide 1 is stabilized by six intramolecular 4 → 1 hydrogen bonds, forming 12 atom C12 motifs. The structure of peptide 2 reveals the formation of eight successive C12 hydrogen-bonded turns. Average backbone dihedral angles for αγ C12 helices are peptide 1, Aib; φ (°) = -57.2 ± 0.8, ψ (°) = -44.5 ± 4.7; γ(4) (R)Ile; φ (°) = -127.3 ± 7.3, θ1 (°) = 58.5 ± 12.1, θ2 (°) = 67.6 ± 10.1, ψ (°) = -126.2 ± 16.1; peptide 2, Aib; φ (°) = -58.8 ± 5.1, ψ (°) = -40.3 ± 5.5; ψ(4) (R)Ile; φ (°) = -123.9 ± 2.7, θ1 (°) = 53.3 θ 4.9, θ 2 (°) = 61.2 ± 1.6, ψ (°) = -121.8 ± 5.1. The tendency of γ(4) -substituted residues to adopt gauche-gauche conformations about the C(α) -C(ß) and C(ß) -C(γ) bonds facilitates helical folding. The αγ C12 helix is a backbone expanded analog of α peptide 310 helix. The hydrogen bond parameters for α peptide 310 and α-helices are compared with those for αγ hybrid C12 helix. Copyright © 2016 European Peptide Society and John Wiley & Sons.

Peptide δ-Turn: Literature Survey and Recent Progress.

Among the various types of α-peptide folding motifs, δ-turn, which requires a central cis-amide disposition, has been one of the least extensively investigated. In particular, this main-chain reversal topology has been studied in-depth neither in linear/cyclic peptides nor in proteins. This Minireview article assembles and critically analyzes relevant data from a literature survey on the δ-turn conformation in those compounds. Unpublished results from recent conformational energy calculations and a preliminary solution-state analysis on a small model peptide, currently ongoing in our laboratories, are also briefly outlined.

C11/C9 helices in crystals of αß hybrid peptides and switching structures between helix types by variation in the α-residue.

Close-packed helices with mixed hydrogen bond directionality are unprecedented in the structural chemistry of α-polypeptides. While NMR studies in solution state provide strong evidence for the occurrence of mixed helices in (ßß)n and (αß)n sequences, limited information is currently available in crystals. The peptide structures presented show the occurrence of C11/C9 helices in (αß)n peptides. Transitions between C11 and C11/C9 helices are observed upon varying the α-amino acid residue.

C12 helices in long hybrid (αγ)n peptides composed entirely of unconstrained residues with proteinogenic side chains.

Unconstrained γ(4) amino acid residues derived by homologation of proteinogenic amino acids facilitate helical folding in hybrid (αγ)n sequences. The C12 helical conformation for the decapeptide, Boc-[Leu-γ(4)(R)Val]5-OMe, is established in crystals by X-ray diffraction. A regular C12 helix is demonstrated by NMR studies of the 18 residue peptide, Boc-[Leu-γ(4)(R)Val]9-OMe, and a designed 16 residue (αγ)n peptide, incorporating variable side chains. Unconstrained (αγ)n peptides show an unexpectedly high propensity for helical folding in long polypeptide sequences.

Conformational analysis of a 20-membered cyclic peptide disulfide from Conus virgo with a WPW segment: evidence for an aromatic-proline sandwich.

A novel peptide containing a single disulfide bond, CIWPWC (Vi804), has been isolated and characterised from the venom of the marine cone snail, Conus virgo. A precursor polypeptide sequence derived from complementary DNA, corresponding to the M-superfamily conotoxins, has been identified. The identity of the synthetic and natural peptide sequence has been established. A detailed analysis of the conformation in solution is reported for Vi804 and a synthetic analogue, CI(D) WPWC ((D) W3-Vi804), in order to establish the structure of the novel WPW motif, which occurs in the context of a 20-membered macrocyclic disulfide. Vi804 exists exclusively in the cis W3P4 conformer in water and methanol, whereas (D) W3-Vi804 occurs exclusively as the trans conformer. NMR spectra revealed a W3P4 type VI ß turn in Vi804 and a type II' ß turn in the analogue peptide, (D) W3-Vi804. The extremely high-field chemical shifts of the proline ring protons, together with specific nuclear Overhauser effects, are used to establish a conformation in which the proline ring is sandwiched between the flanking Trp residues, which emphasises a stabilising role for the aromatic-proline interactions, mediated predominantly by dispersion forces.

Conformational diversity in contryphans from Conus venom: cis-trans isomerisation and aromatic/proline interactions in the 23-membered ring of a 7-residue peptide disulfide loop.

Conformational diversity or "shapeshifting" in cyclic peptide natural products can, in principle, confer a single molecular entity with the property of binding to multiple receptors. Conformational equilibria have been probed in the contryphans, which are peptides derived from Conus venom possessing a 23-membered cyclic disulfide moiety. The natural sequences derived from Conus inscriptus, GCV(D)LYPWC* (In936) and Conus loroisii, GCP(D)WDPWC* (Lo959) differ in the number of proline residues within the macrocyclic ring. Structural characterisation of distinct conformational states arising from cis-trans equilibria about Xxx-Pro bonds is reported. Isomerisation about the C2-P3 bond is observed in the case of Lo959 and about the Y5-P6 bond in In936. Evidence is presented for as many as four distinct species in the case of the synthetic analogue V3P In936. The Tyr-Pro-Trp segment in In936 is characterised by distinct sidechain orientations as a consequence of aromatic/proline interactions as evidenced by specific sidechain-sidechain nuclear Overhauser effects and ring current shifted proton chemical shifts. Molecular dynamics simulations suggest that Tyr5 and Trp7 sidechain conformations are correlated and depend on the geometry of the Xxx-Pro bond. Thermodynamic parameters are derived for the cis↔trans equilibrium for In936. Studies on synthetic analogues provide insights into the role of sequence effects in modulating isomerisation about Xxx-Pro bonds.

Unconstrained homooligomeric γ-peptides show high propensity for C14 helix formation.

Monosubstituted γ(4)-residues (γ(4)Leu, γ(4)Ile, and γ(4)Val) form helices even in short homooligomeric sequences. C14 helix formation is established by X-ray diffraction in homooligomeric (γ)n tetra-, hexa- and decapeptide sequences demonstrating the high propensity of γ residues, with proteinogenic side chains, to adopt locally folded conformations.

Analysis of designed ß-hairpin peptides: molecular conformation and packing in crystals.

The crystal structures of several designed peptide hairpins have been determined in order to establish features of molecular conformations and modes of aggregation in the crystals. Hairpin formation has been induced using a centrally positioned (D)Pro-Xxx segment (Xxx = (L)Pro, Aib, Ac6c, Ala; Aib = α-aminoisobutyric acid; Ac6c = 1-aminocyclohexane-1-carboxylic acid). Structures of the peptides Boc-Leu-Phe-Val-(D)Pro-(L)Pro-Leu-Phe-Val-OMe (1), Boc-Leu-Tyr-Val-(D)Pro-(L)Pro-Leu-Phe-Val-OMe (2, polymorphic forms labeled as 2a and 2b), Boc-Leu-Val-Val-(D)Pro-(L)Pro-Leu-Val-Val-OMe (3), Boc-Leu-Phe-Val-(D)Pro-Aib-Leu-Phe-Val-OMe (4, polymorphic forms labeled as 4a and 4b), Boc-Leu-Phe-Val-(D)Pro-Ac6c-Leu-Phe-Val-OMe (5) and Boc-Leu-Phe-Val-(D)Pro-Ala-Leu-Phe-Val-OMe (6) are described. All the octapeptides adopt type II' ß-turn nucleated hairpins, stabilized by three or four cross-strand intramolecular hydrogen bonds. The angle of twist between the two antiparallel strands lies in the range of -9.8° to -26.7°. A detailed analysis of packing motifs in peptide hairpin crystals is presented, revealing three broad modes of association: parallel packing, antiparallel packing and orthogonal packing. An attempt to correlate aggregation modes in solution with observed packing motifs in crystals has been made by indexing of crystal faces in the case of three of the peptide hairpins. The observed modes of hairpin aggregation may be of relevance in modeling multiple modes of association, which may provide insights into the structure of insoluble polypeptide aggregates.

A designed three-stranded ß-sheet in an α/ß hybrid peptide.

The incorporation of ß-amino acid residues into the antiparallel ß-strand segments of a multi-stranded ß-sheet peptide is demonstrated for a 19-residue peptide, Boc-LV(ß)FV(D)PGL(ß)FVVL(D)PGLVL(ß)FVV-OMe (BBH19). Two centrally positioned (D)Pro-Gly segments facilitate formation of a stable three-stranded ß-sheet, in which ß-phenylalanine ((ß)Phe) residues occur at facing positions 3, 8 and 17. Structure determination in methanol solution is accomplished by using NMR-derived restraints obtained from NOEs, temperature dependence of amide NH chemical shifts, rates of H/D exchange of amide protons and vicinal coupling constants. The data are consistent with a conformationally well-defined three-stranded ß-sheet structure in solution. Cross-strand interactions between (ß)Phe3/(ß)Phe17 and (ß)Phe3/Val15 residues define orientations of these side-chains. The observation of close contact distances between the side-chains on the N- and C-terminal strands of the three-stranded ß-sheet provides strong support for the designed structure. Evidence is presented for multiple side-chain conformations from an analysis of NOE data. An unusual observation of the disappearance of the Gly NH resonances upon prolonged storage in methanol is rationalised on the basis of a slow aggregation step, resulting in stacking of three-stranded ß-sheet structures, which in turn influences the conformational interconversion between type I' and type II' ß-turns at the two (D)Pro-Gly segments. Experimental evidence for these processes is presented. The decapeptide fragment Boc-LV(ß)FV(D)PGL(ß)FVV-OMe (BBH10), which has been previously characterized as a type I' ß-turn nucleated hairpin, is shown to favour a type II' ß-turn conformation in solution, supporting the occurrence of conformational interconversion at the turn segments in these hairpin and sheet structures.

Rapid mass spectrometric determination of disulfide connectivity in peptides and proteins.

Disulfide crosslinks are ubiquitous in natural peptides and proteins, providing rigidity to polypeptide scaffolds. The assignment of disulfide connectivity in multiple crosslinked systems is often difficult to achieve. Here, we show that rapid unambiguous characterisation of disulfide connectivity can be achieved through direct mass spectrometric CID fragmentation of the disulfide intact polypeptides. The method requires a direct mass spectrometric fragmentation of the native disulfide bonded polypeptides and subsequent analysis using a newly developed program, DisConnect. Technical difficulties involving direct fragmentation of proteins are surmounted by an initial proteolytic nick and subsequent determination of the structures of these proteolytic peptides through DisConnect. While the connectivity in proteolytic fragments containing one cystine is evident from the MS profile alone, those with multiple cystines are subjected to subsequent mass spectrometric fragmentation. The wide applicability of this method is illustrated using examples of peptide hormones, peptide toxins, proteins, and disulfide foldamers of a synthetic analogue of a marine peptide toxin. The method, coupled with DisConnect, provides an unambiguous, straightforward approach, especially useful for the rapid screening of the disulfide crosslink fidelity in recombinant proteins, determination of disulfide linkages in natural peptide toxins and characterization of folding intermediates encountered in oxidative folding pathways.