Capillary electrophoresis coupled with mass spectrometry for the evaluation of substance P enzymatic degradation by SaOS-2 human osteosarcoma.

A new analytical method for the detection and the quantitative evaluation of the undecapeptide substance P by capillary electrophoresis coupled with ion trap mass spectrometry (CE-MS) by a co-axial sheath liquid interface has been developed. Conditions of analysis employed an acidic buffer and a 60 cm fused silica capillary installed by overcoming the UV window position, thus allowing to perform the analysis in a brief time. The method has been applied to the evaluation of substance P enzymatic hydrolysis during incubation with the human osteosarcoma SaOS-2 cell line. The analysis of amino acids derived from the cleavage of substance P has been also carried out simultaneously under the same electrophoretic conditions allowing the description of a kinetic of amino acid formation, parallel with substance P disappearance. The amounts of intact substance P and of free amino acids were monitored along 600 s of incubation time. A steady decrease of substance P as function of reaction time was observed. Peptide's half-life was found to be about 4.3s, indicating an extremely fast hydrolysis in the presence of the SaOS-2 cells. Proline, phenilalanine and methionine were the predominant free amino acids recorded. Obtained results lead to hypothesize the occurrence of endopeptidases activity, followed by aminopeptidases responsible for the release of free amino acids originated after primary bond cleavage.