Objectives: the aim of this study was the development of techniques deriving from capillary electrophoresis (CE), which can be utilized for the analysis of salivary proteins. Methods: the CE apparatus utilized were either a P/ACE MDQ system equipped by diode array detector or a P/ACE 2100 system. Separation of whole salivary samples performed by classical zonal CE in acidic buffer (80 mmol/l sodium phosphate buffer, pH 2.65) did not provided high resolution results, even with a use of a capillary coated by a polyacrylamide monolayer. Results: several peaks pertaining to basic and acidic proline-rich proteins (PRP) are detectable, but neither histatins, nor statherin, nor cystatins were detectable, probably due to their low concentration and their high interaction with the inner capillary wall. When the separation is performed in neutral environment using sodium dodecyl sulphate (SDS) and entangled polymers, therefore according to protein dimensions, several peaks pertaining to histatins, PRP, amylases and mucins are detectable. When the CE separation is performed in basic buffer and SDS (20 mmol/l TAPS; SDS 70 mmol/l, pH 9.3 buffer, mixed in a ratio 70/30 with acetonitrile), hence in micellar electrokinetic conditions, the resolution increased and several peaks pertaining to histatin and acidic and basic PRP are detectable, whereas statherin is not detectable. Finally, when the separation is performed in micellar conditions, using an acidic environment (80 mmol/l sodium phosphate, 50 mmol/l SDS, pH 2.70) and reversed polarity, the order of migration appeared inverted with respect to the usual RP-HPLC separations of salivary peptides. The migration order appeared to be acidic PRP, basic PRP and, in high load sample conditions, several peaks probably pertaining to histatins are detectable. Conclusions: none of the different options tested is able to provide a comprehensive picture of different human salivary classes and all the options were characterized by scarce reproducibility and stability.
Different capillary electrophoresis options for the analysis of salivary proteins
MESSANA, IRENE;
2002-01-01
Abstract
Objectives: the aim of this study was the development of techniques deriving from capillary electrophoresis (CE), which can be utilized for the analysis of salivary proteins. Methods: the CE apparatus utilized were either a P/ACE MDQ system equipped by diode array detector or a P/ACE 2100 system. Separation of whole salivary samples performed by classical zonal CE in acidic buffer (80 mmol/l sodium phosphate buffer, pH 2.65) did not provided high resolution results, even with a use of a capillary coated by a polyacrylamide monolayer. Results: several peaks pertaining to basic and acidic proline-rich proteins (PRP) are detectable, but neither histatins, nor statherin, nor cystatins were detectable, probably due to their low concentration and their high interaction with the inner capillary wall. When the separation is performed in neutral environment using sodium dodecyl sulphate (SDS) and entangled polymers, therefore according to protein dimensions, several peaks pertaining to histatins, PRP, amylases and mucins are detectable. When the CE separation is performed in basic buffer and SDS (20 mmol/l TAPS; SDS 70 mmol/l, pH 9.3 buffer, mixed in a ratio 70/30 with acetonitrile), hence in micellar electrokinetic conditions, the resolution increased and several peaks pertaining to histatin and acidic and basic PRP are detectable, whereas statherin is not detectable. Finally, when the separation is performed in micellar conditions, using an acidic environment (80 mmol/l sodium phosphate, 50 mmol/l SDS, pH 2.70) and reversed polarity, the order of migration appeared inverted with respect to the usual RP-HPLC separations of salivary peptides. The migration order appeared to be acidic PRP, basic PRP and, in high load sample conditions, several peaks probably pertaining to histatins are detectable. Conclusions: none of the different options tested is able to provide a comprehensive picture of different human salivary classes and all the options were characterized by scarce reproducibility and stability.
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