RESUMO
RATIONALE: Mass spectrometry has shown itself as 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 useful methods of triggering fragmentation and combine complementary techniques. METHODS: Comparison of low-energy collision-induced dissociation (CID) and higher energy collision-induced dissociation (HCD) modes for sequencing of the natural non-tryptic peptides with disulfide bonds and/or several proline residues in the backbone was achieved using an LTQ FT Ultra Fourier transform ion cyclotron resonance (FTICR) mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with a 7 T magnet and an LTQ Orbitrap Velos ETD (Thermo Fisher Scientific, Bremen, Germany) instrument. Peptide fractions were obtained by high-performance liquid chromatography (HPLC) separation of frog skin secretion samples from ten species of Rana temporaria, caught in the Kolomna district of Moscow region (Russia). RESULTS: HCD makes the b/y series longer and more pronounced, thus increasing sequence coverage. Fragment ions due to cleavages at the C-termini of proline residues make the sequencing more reliable and may be used to detect missed cleavages in the case of tryptic peptides. Another HCD peculiarity involves formation of pronounced inner fragment ions (secondary y(n)b(m) ion series formed from the abundant primary y-ions). Differences in de novo sequencing of natural non-tryptic peptides with CID and HCD, involving thorough manual expert interpretation of spectra and two automatic sequencing algorithms, are discussed. CONCLUSIONS: Although HCD provides better results, a combination of CID and HCD data may notably increase reliability of de novo sequencing. Several pairs of b2 /a2 -ions may be formed in HCD, complicating the spectra. Automatic de novo sequencing with the available programs remains less efficient than the manual one, independently of the collision energy.
Assuntos
Espectrometria de Massas/métodos , Peptídeos/análise , Peptídeos/química , Análise de Sequência de Proteína/métodos , Sequência de Aminoácidos , Animais , Rana temporaria , Pele/químicaRESUMO
Long disulphide-containing peptides brevinins 1E and 2Ec from the skin secretion of the frog Rana ridibunda were reduced and alkylated with ten novel and three known derivatizing agents. Nine of novel reagents are maleimide derivatives. The peptides were also reduced with DTT directly onto the MALDI target without alkylation. Modified samples were subjected to MALDI-PSD study. Procedures, fragmentation patterns, fragment ion signal abundances and sequence coverage for two peptides modified with thirteen tags (or on-plate reduced) are described. The fast on-plate procedure for reduction/alkylation was applied to Rana ridibunda crude secretion, providing intensive signals of derivatized peptides. The corresponding ions may be used for the MS/MS sequencing procedure.
Assuntos
Proteínas de Anfíbios/química , Peptídeos Catiônicos Antimicrobianos/química , Cistina/química , Rana ridibunda , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Sequência de Aminoácidos , Animais , Dissulfetos/química , Feminino , Masculino , Técnicas de Sonda Molecular , Dados de Sequência MolecularRESUMO
Identification of species constituting Rana esculenta complex represents a certain problem as two parental species Rana ridibunda and Rana lessonae form their hybrid R. esculenta, while external signs and sizes of the members of this complex are intersected. However the composition of skin secretion consisting mainly of peptides is different for the species of the complex. LC-MS/MS is an ideal analytical tool for the quantitative and qualitative analysis of these peptides. The results covering elemental composition of these peptides, their levels in the secretion, as well as their belonging to a certain family of peptides may be visualized by means of 2D mass maps. The proposed approach proved itself to be a perspective tool for the reliable identification of all 3 species constituting R. esculenta complex. Easy distinguishing between the species may be achieved using 2D maps as fingerprints. Besides this approach may be used to study hybridogenesis and mechanisms of hemiclonal transfer of genetic information, when rapid and reliable identification of species involved in the process is required.