EThcD Discrimination of Isomeric Leucine/Isoleucine Residues in Sequencing of the Intact Skin Frog Peptides with Intramolecular Disulfide Bond.

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. MS3 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.

Differentiation of frogs from two populations belonging to the Pelophylax esculentus complex by LC-MS/MS comparison of their skin peptidomes.

LC-MS/MS was applied to establish the composition of the skin peptidome of a Slovenian green frog belonging to the Pelophylax esculentus complex. As this was similar to the peptidome of the Moscow population of Pelophylax ridibundus, it allowed us to identify the Slovenian frog from the Pelophylax esculentus complex as Pelophylax ridibundus. The sequences of six new peptides from the brevinin 2 family are reported for the first time on the basis of manual interpretation of their tandem mass spectra. The structural similarity of the brevinin 2 peptides from the Moscow and Slovenian populations of Pelophylax ridibundus enables peptides from this family to be utilized as biomarkers for Pelophylax ridibundus inter- and intraspecies differentiation, and the proposed approach can be used as an analytical tool for differentiating the corresponding species and populations. The potential biological activities of the novel peptides were estimated by 2D mass mapping. The results allowed us to classify all of the available peptides belonging to the brevinin 2 family. Graphical Abstract Intraspecies identification within the green frog complex.

LTQ Orbitrap Velos in routine de novo sequencing of non-tryptic skin peptides from the frog Rana latastei with traditional and reliable manual spectra interpretation.

RATIONALE: Mass spectrometry has shown itself to be the most efficient tool for the sequencing of peptides. However, de novo sequencing of novel natural peptides is significantly more challenging in comparison with the same procedure applied for the tryptic peptides. To reach the goal in this case it is essential to select the most efficient methods of triggering fragmentation and combine all the possible complementary techniques. METHODS: Collision-induced dissociation (CID), high-energy collision dissociation (HCD), and electron-transfer dissociation (ETD) tandem mass spectra recorded with a LTQ Orbitrap Velos instrument were used for the elucidation of the sequence of the natural non-tryptic peptides from the skin secretion of Rana latastei. Manual interpretation of the spectra was applied. RESULTS: The combined approach using CID, HCD, and ETD tandem mass spectra of the multiprotonated peptides in various charge states, as well as of their proteolytic fragments, allowed the sequences of seven novel peptides from the skin secretion of Rana latastei to be established. CONCLUSIONS: Manual mass spectrometry sequencing of natural non-tryptic peptides from the skin secretion of Rana latastei provided the opportunity to work successfully with these species and demonstrated once again its advantage over automatic approaches.