Mass Spectrometry Differentiation between Rana arvalis Populations Based on Their Skin Peptidome Composition

Abstract

Skin secretion of amphibians often represents the only weapon of these species against pathogens and predators. Peptides constitute the major portion of active molecules of that weapon and may be treated as potential pharmaceuticals for future generations. The first step of their efficient use involves establishing of their primary structure, i.e., sequencing. De novo sequencing by means of mass spectrometry was applied to Rana arvalis species, collected in the spring 2021 in Central Slovenia (vicinity of Ljubljana). HPLC-ESI-HRMS/MS with Orbitrap instruments was used to establish the skin peptidome of these species and compare it with the earlier identified skin peptidome of the Moscow population of Rana arvalis. Application of CID, HCD, ETD, and EThcD enabled detecting and sequencing 18 peptides; five of them were novel and may be treated as possible biomarkers of the Ljubljana population of Rana arvalis. Interestingly, representatives of two peptide families (temporins and brevinins 2) were not found in the Moscow population. MS3 modes, first of all EThcD, demonstrated their great potential in the de novo sequencing, including extraction of the sequence information from the intact peptides with disulfide cycle (rana box) in their structure and differentiation of isomeric Leu/Ile residues. Thus, all six isomeric residues were reliably distinguished in the novel melittin-related peptide AK-23-1. In addition, another post-translational modification dealing with carbonylation of the N-terminal Gly of novel temporin AVa was established using the MS3 mode. The obtained results demonstrate the efficiency of the use of MS3 tools in proteomics/peptidomics.

EThcD Benefits for the Sequencing Inside Intramolecular Disulfide Cycles of Amphibian Intact Peptides

“…Table 1 lists S−S-bond-containing peptides detected and sequenced in the secretion of Rana arvalis, Rana temporaria, and Pelophylax esculentus frogs. 15,56,58,59 The table provides the full sequence of the peptides and the portion of intact disulfide cycle established by means of HCD and EThcD spectra. The efficiency of the approach is dependent on the peptide length and the sequence inside the loop.…”

“…Continuing our studies of frog peptides, we have applied the EThcD MS 3 method to the intact peptides, first of all to distinguish between the isomeric Leu/Ile residues. , However, besides the expected w-ions, we noticed a series of c-ions related to the sequence inside the cyclic portion of the analyzed peptides’ structure. Table lists S–S-bond-containing peptides detected and sequenced in the secretion of Rana arvalis, Rana temporaria, and Pelophylax esculentus frogs. ,,, The table provides the full sequence of the peptides and the portion of intact disulfide cycle established by means of HCD and EThcD spectra. The efficiency of the approach is dependent on the peptide length and the sequence inside the loop.…”

“…All EThcD tandem experiments were performed using an Orbitrap Fusion mass spectrometer (Thermo Fischer Scientific) in HPLC/MS/MS mode as previously described. ,, Chromatographic separation was performed using a 50 cm long EASY spray column. Gradient parameters were as follows: 4% (B) for 7 min, 4–50% (B) in 21 min, 50–80% (B) in 5 min, 80% (B) for 5 min, 95% (B) and 95–4% (B) in 5 min.…”

EThcD as a Unique Tool for the Top-Down De Novo Sequencing of Intact Natural Ranid Amphibian Peptides

“…All EThcD experiments were performed using an Orbitrap Fusion mass spectrometer (Thermo Fischer Scientific) as previously described. ,, Chromatographic separation was performed using a 50 cm-long EASY nanoflow column. Gradient parameters were as follows: 4% (B) for 7 min, 4–50% (B) in 21 min, 50–80% (B) in 5 min, 80% (B) for 5 min, and 95% (B) and 95–4% (B) in 5 min.…”

Temporins: Multifunctional Peptides from Frog Skin

“…The diversity of secreted peptides was hypothesized to be a strategy to gain maximum protection from different microorganisms and reduce the chance of inducing resistance to individual peptides [ 7 ]. The pattern of secreted peptides can be employed as a biomarker of a particular frog population [ 8 ]. In some species, AMP production is regulated by the thyroid hormones, and may be seasonal and affected by environmental factors [ 9 , 10 ].…”