Characterization of antimalarial SPf66 peptide using MALDI-TOF MS, CD and SEC

Dpto. Ingeniería Química y Tecnología Farmacéutica, Facultad de Farmacia, Universidad de La Laguna, Tenerife, Spain.
Peptides (Impact Factor: 2.62). 10/2002; 23(9):1527-35. DOI: 10.1016/S0196-9781(02)00093-1
Source: PubMed


SPf66 is the first chemically synthesized peptide to elicit a partial protective immune response against malaria. Size-exclusion chromatography (SEC) with multi-angle laser light-scattering (MALLS) detection and hydrogen/deuterium (H/D) exchange monitored by (matrix-assisted laser desorption/ionization) MALDI-TOF (time-of-flight) mass spectrometry (MS) were used to assess the conformation and stability in aqueous solution after storage at different temperatures. Moreover, the feasible conformational changes of this peptide were also measured by circular dichroism (CD)-spectroscopy. The absolute molecular weight of SPf66 monomer and dimer species were 4765 and 8960Da using SEC with MALLS detection, and 4643 and 9490Da by MALDI-TOF MS, the discrepancy being between both methods lower than 5.7%, a value quite close to those found in other proteins. The results from H/D exchange monitored by MALDI-TOF MS and CD-spectroscopy show that the SPf66 monomer lacks ordered structure, whereas the SPf66 dimer species presents segments of secondary structure as a determined by CD measurements.

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    ABSTRACT: Stability studies on the SPf66 antimalarial peptide with different pH and temperature conditions were carried out. The degradation mechanism was elucidated by the size-exclusion chromatography (SEC) technique and the experimental data obtained at 37 degrees C and different pH were fitted to a kinetic degradation model that could explain the loss of its immunogenic capacity. At 5, 25, 37, and 70 degrees C and pH 2, changes were detected in the areas of the different species, although the values obtained could not be fitted to any known degradation kinetics.
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    ABSTRACT: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used in hydrogen exchange studies, exchanging deuteron (H/D) or proton (D/H), to determine the structure and conformational changes of antimalarial Spf66 synthetic peptide in its monomeric and dimeric forms. The accuracy of both analytical methods was assessed along with their suitability to study structural aspects. The results via these two approaches were in agreement, indicating that the dimer presents segments of secondary structure. In this last case, the combination of both methods with enzymatic digestion with pepsin was used in their identification. Although 100% coverage of Spf66 dimer was not observed, the higher levels of deuteration were observed for fragments located in the chain terminal where the structure may be more flexible, while the fragments near the disulfide bonds, which is, in theory, the more rigid region of the molecule, were not detected. This strategy is significantly time saving and allows rapid screening and help to characterize a protein, especially, when no prior structural information is available. However, a single spectrum is not certainly sufficient to obtain structural data; it is just an experimental limitation. Also, changes in peptide structure after storage at different temperatures and time were observed, which lead to a loss in the secondary structure as determined by circular dicroism measurements and an increase in aggregation products, since the trimer and tetramer species were detected by mass spectrometry.
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