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Our scientific interests involve de novo sequencing of non-tryptic natural amphibian skin peptides including those with intramolecular S-S bond by means of exclusively mass spectrometry. Reliable discrimination of the isomeric leucine/isoleucine residues during peptide sequencing by means of mass spectrometry represents a bottleneck in the workflow for complete automation of the primary structure elucidation of these compounds. MS 3 is capable of solving the problem. Earlier we demonstrated the advanced efficiency of ETD-HCD method to discriminate Leu/Ile in individual peptides by consecutive application of ETD to the polyprotonated peptides followed by HCD applied to the manually selected primary z-ions with the targeted isomeric residues at their N-termini and registration of the characteristic w-ions. Later this approach was extended to deal with several (4-7) broad band mass ranges, without special isolation of the primary z-ions. The present paper demonstrates an advanced version of this method when EThcD is applied in the whole mass range to a complex mixture of natural non-tryptic peptides without their separation and intermediate isolation of the targeted z-ions. The proposed EThcD method showed over 81% efficiency for the large natural peptides with intact disulfide ring, while the interfering process of radical site migration is suppressed. Due to higher speed and sensitivity, the proposed EThcD approach facilitates the analytical procedure and allows for the automation of the entire experiment and data processing. Moreover, in some cases it gives a chance to establish the nature of the residues in the intact intramolecular disulfide loops. Graphical Abstract ᅟ.

Electron-transfer dissociation (ETD) and electron-transfer and higher-energy collision dissociation (EThcD) spectra of short tryptic peptides with leucine/isoleucine residues in neighboring positions demonstrate intensive w-ions. On the contrary, u-ions possess very low intensities (if present at all). Therefore radical site migration is negligible in the applied conditions while ETD (EThcD) spectra allow for the reliable discrimination of the isomeric residues in the sequencing process. The presence of a fragment ion 43.055 mass units lower than z 2 -ion of peptides with IK sequence at their C-termini was shown to be a result of alternative fragmentation starting from the loss of propylammonium ion from the doubly protonated peptide molecule and formation of an oxazole fragment ion.

Characterization and differentiation of isomers in biological macromolecules using mass spectrometry is one of the most significant challenges facing scientists in the field. The capability of high-resolution MS instruments along with the development of new fragmentation methods now provides the ability to indirectly differentiate between some isomers. This ability has enabled mass spectrometry to evolve into a multidisciplinary technique incorporating areas such as pharmaceutical research, proteomics, polymer science, medicine, environmental chemistry, and recently archeology. This article aims to review recent developments in mass spectrometry methodologies in the identification of structural and spatial isomers in biological macromolecules, such as aspartic acid and isoaspartic acid (Asp/IsoAsp), leucine and isoleucine (Leu/Ile), glutamic acid and γ-glutamic acid, and D/L enantiomers., (Copyright © 2012 Wiley Periodicals, Inc.)

Collisional cooling employed in an orthogonal time-of-flight mass spectrometer (o-TOF MS) stabilizes fragment ions that are generated by matrix-assisted laser desorption ionization in-source decay (MALDI ISD). By variation of the buffer gas pressure, ISD and "post-source decay" (PSD) dissociation channels can be switched on and off to some extent. Under high-pressure conditions, ISD type fragments of post-translationally modified (PTM) peptides are generated that contain even labile bound side groups; the examples of a phosphorylated and an O-glycosylated peptide are shown. At elevated laser fluences, d- and w-type fragment ions of peptides are detected as a result of high-energy side chain cleavage. This allows for differentiation of the isobaric amino acid residues leucine and isoleucine. Reduction of the cooling efficiency by lowering the buffer gas pressure results in the loss of the d-/w-type species, presumably in secondary metastable dissociation processes. This also enhances cleavage of the side groups from the PTM peptides and can be used to corroborate identification of the modification site. Furthermore, these measuring conditions generate small amounts of fragments containing sequence information about the cyclic part of a disulfide peptide by inducing symmetric and asymmetric cleavage of the intramolecular S-S bond.

Tripeptides form ternary complexes with Cu(2+) and 2,2'-bipyridine (bpy) that self-assemble upon mixing the components in aqueous methanol solution. Electrospray ionization (ESI) of the complex solutions provides abundant singly charged [Cu(peptide -- H)bpy](+) and doubly charged [Cu(peptide)bpy](2+) ions. Collision-induced dissociation (CID) at low ion kinetic energies of several tripeptides, AGG, GGA, LGG, GGL, GGI, FGG, GGF, LGF, GLF, GFL, GYA and GAY, showed fragments that were indicative of the amino acid sequence in the peptide. In addition, CID of single and doubly charged complexes of isomeric tripeptides GGL and GGI provided unambiguous distinction of the isomeric leucine and isoleucine residues. Leucine peptides eliminated C(3)H(7) radicals from the amino acid side-chain whereas isoleucine eliminated C(2)H(5) radicals. CID of gas-phase doubly charged peptide complexes in a quadrupole ion trap produced a series of singly charged sequence fragments that following isolation and further CID furnished distinct fragments that allowed quantitation of leucine and isoleucine-containing peptides in mixtures., (Copyright 2001 John Wiley & Sons, Ltd.)

Antimicrobial peptides are widely distributed in living organisms, where they represent a constitutive defence system acting as a first line of response against infections. The number of such peptides discovered has increased rapidly in the last few years, and more than 100 have been described from different sources. So far, antimicrobial peptides containing a D-amino acid have only been found in the skin secretions of frogs belonging to the genus Bombina. In the second position of the sequence of the mature peptides either D-alloisoleucine or D-leucine were detected. The D-amino acids are derived from the corresponding L forms by an as yet unknown posttranslational reaction.

Phenylthiohydantoin (PTH) amino acid derivatives are formed during sequential Edman degradation of peptides and proteins. Isomeric Leu and Ile (which differ only in respectively having iso-butyl and sec-butyl chains, and are often difficult to distinguish by conventional mass spectrometric techniques) may be readily identified by the characteristic decompositions of the [M-H]- ions of their PTH derivatives. The Leu spectrum shows major loss of propane, while that of the Ile derivative shows elimination of both methane and ethane. This method may be used routinely with 10 microgram quantities of peptide material.

A family of bombinin-related peptides is present in the skin of Bombina variegata. These peptides contain 27 residues with Gly as N-terminus and display antimicrobial activity. From sequence analysis of the cDNAs encoding for the corresponding peptide precursors, the presence of a novel 20-residue peptide with Ile as N-terminus was predicted. We have now purified a family of hydrophobic peptides named H1-H5, whose sequences correspond to the predicted peptide with some variability in positions 1, 2 and 8. In particular, H3-H5 contain a D-alloisoleucine residue in the second position. All these peptides display antibacterial and haemolytic activity.

A diagonal-electrophoresis method for the selective purification of serine phosphate peptides was applied to tryptic, chymotryptic and peptic digests of oxidized ovalbumin. This method is based on the release of the phosphate group bound to serine by treatment with alkaline phosphatase on paper. The identified serine phosphate peptides were purified by paper electrophoresis at pH6.5 and 2.0, dephosphorylation with bacterial alkaline phosphatase, and paper electrophoresis at pH2.0 again, in that order. The presence of two groups of serine phosphate peptides was apparent from the amino acid composition. One group contained no lysine, cysteic acid, proline, leucine or isoleucine (sequence 1) and the other had all those amino acids (sequence 2). Further degradation with subtilisin of those peptides and ;dansyl'-Edman sequence analysis established their partial sequences. The proposed sequences are as follows (with ;SerP' representing serine phosphate): sequence 1, -Ala-Gly-Arg-Glu-Val-Val-Gly-SerP-Ala-Glu-Ala-Gly-Asp-Val-Ala-Ala-Ser-(Val,Glx(2),Ser,Phe)-Arg-; sequence 2, -Asp-Lys-Leu-Pro-Gly-Phe-Gly-Asp-SerP-Ile-Glx-Ala-Glx-CySO(3)H-Gly-(Thr,Ser,Val)-(Asp,His,Val)-. The partial sequence of one of the phosphopeptides, Asp-(Glu,Ile,SerP), reported by Flavin (1954) was used to establish the proposed sequence 2.

Freely occurring alpha-l-aspartyl-l-histidine, beta-alanyl-l-histidine (carnosine), alpha-l-aspartyl-alpha-l-lysine, l-asparaginyl-alpha-l-lysine, alpha-l-glutamyl-alpha-l-lysine, l-isoleucyl-alpha-l-lysine, l-seryl-alpha-l-lysine, l-valyl-alpha-l-lysine and glycyl-alpha-l-lysyl-alpha-l-lysine were identified in young larvae (second and third instars) of the blowfly (Phormia regina).