Analysis of sulfated peptides from the skin secretion of the Pachymedusa dacnicolor frog using IMAC‐Ga enrichment and high‐resolution mass spectrometry

Abstract

Immobilized metal ion affinity chromatography (IMAC) has been widely used for the enrichment of phosphopeptides, whereas no report exists describing the use of IMAC columns for the enrichment of sulfopeptides. In this study, we used IMAC-Ga microcolumns for the enrichment of sulfopeptides from a complex mixture of peptides, extracted from skin secretions of the Pachymedusa dacnicolor frog. The enriched fraction obtained by IMAC-Ga was analyzed by liquid chromatograpy/electrospray ionization mass spectrometry (LC/ESI-MS) in an Orbitrap XL and by matrix-assisted laser desorption/ionization time-of-flight time-of-flight (MALDI-TOF/TOF) in an ABI 4800 instrument. From this fraction, different sulfated and non-sulfated peptides belonging to the caerulin and bradykinin families were structurally characterized. Other interesting negatively charged groups, such as phosphate adducts of dermaseptins and pyridoxal phosphate attached to a protease inhibitor, were also characterized. Unexpectedly, some dermaseptin antimicrobial peptides were also enriched by IMAC-Ga and a Sauvatine-like peptide was also fully sequenced. Furthermore, neutral loss of sulfated peptides and their fragmentation patterns in the gas phase were also compared using collision-induced dissociation (CID) and high-energy collision dissociation (HCD). Our present study provides evidence that IMAC-Ga enrichment is a fast, useful and promising method for high-throughput analysis of sulfated-peptides, since high-resolution mass spectrometers can be used for this purpose.

Direct Identification of Tyrosine Sulfation by using Ultraviolet Photodissociation Mass Spectrometry

“…For a complex biological sample, enrichment of sulfated peptides would be necessary based on their low stoichiometric abundances relative to unmodified proteins in the sample. Various enrichment methods have been reported including ones that utilize immobilized metal affinity chromatography, weak anion exchange, and anti-sulfotyrosine monoclonal antibodies and we are currently integrating a robust enrichment method with the sensitive UVPD-MS approach[60–62]. Additionally, the UVPD method is not designed for direct peptide quantification per se, although this could be achieved using peptide standards to generate a calibration curve or through the use of various label or label-free methods for relative quantification between samples.…”

A Review on Bradykinin-Related Peptides Isolated from Amphibian Skin Secretion

“…The unique kind of peptide in the skin secretion of sub-family Phyllomedusinae is phyllokinin (PK), which displays two amino acid residues Ile-Tyr extended at C -terminals. PK was first reported from the studies of skin of Phyllomedusa rohdei with a post-translational modification of tyrosine O -sulfation and this peptide was subsequently discovered from other species in this sub-family [ 29 , 32 , 33 , 35 , 41 , 42 ]. Similar to PK, some of the BRPs were C -terminal extended with one or two amino acid residues, which are considered to be more potent than conventional BK because the extended region could inhibit the enzymatic metabolism of degradation of the BRPs, which prolong the ligand-receptor interaction [ 43 ].…”

“…Apparently, there is a special sulfatase widely distributed in the Phyllomedusinae tree frogs, which contributes to the generation of PK in the skin secretion for the purpose of self-defence. In the process of review, there is a non-uniform nomenclature, which is causing confusion between “PK” and “PK sulfated” in some cases [ 29 , 30 , 34 , 42 ]. Considered in the first publication of PK, which is a sulfated form in skin secretion, it should be named the Tyr residue O -sulfated form of PK in future studies in order to avoid any misunderstanding.…”

Advances in purification and separation of posttranslationally modified proteins

“…For example, immobilized Ga ions can separate sulfated peptides [34] and several combinations of metal ions and metal oxides are widely used for phosphoproteome analysis (see 3.1.2.). Naturally occurring PTMs often participate in cell signaling and proteinprotein interactions.…”