Micropreparative separation, fractionation, and peptide mapping of beta-lactoglobulin A and B variants by capillary electrophoresis.
The methodological aspects for the separation, fractionation, and peptide mapping by free zone capillary electrophoresis (CZE) of beta-lactoglobulin (beta-Lg) variants A and B were established. First, beta-Lg variants A or B were separated and fractionated by CZE. Then, the collected protein fraction was subjected to off-line tryptic digestion. Second, peptide mapping of the tryptic hydrolysates and peptide fraction collection were carried out by CZE. beta-Lg variants were separated and collected using an uncoated capillary (72 cm x 75 microm i.d.) in 0.05 M borate buffer containing 0.1% Tween 20 at pH 8.0 by applying 20 kV. By subjecting the capillary under pressure after a delay time of 15%, the protein was collected in a microvial containing digestion buffer. The most suitable conditions for the tryptic digestion of beta-Lg were established by monitoring the reaction products with CZE. A tryptic hydrolysis with an enzyme-to-substrate ratio (E/S) of 1/20 and incubation for 20 hr at 37 degrees C was found to result in the most suitable conditions. Peptides were separated and collected using an uncoated capillary (120 cm x 75 microm i.d.) in 0.15 M formic acid at pH 2.3 by applying 28 kV. Peptide maps were highly reproducible as shown by coefficients of variation of less than 0.89 and 5.42% for migration times and peak areas, respectively. Moreover, very good resolution of the peptide maps revealed the region in which the aberrant peptides of the beta-Lg variants may be located.
Olguin-Arredondo
Hector Armando
HA
Centro de Investigación en Alimentaciń y Desarrollo, A. C., Carretera a La Victoria, Hermosillo, Sonora, México.
Vallejo-Cordoba
Belinda
B
González-Córdova
Aarón Fernando
AF
D005591:Chemical Fractionation / Q000379:methods*; D019075:Electrophoresis, Capillary / Q000379:methods*; D007782:Lactoglobulins / Q000032:analysis* / Q000737:chemistry* / Q000302:isolation & purification; D008832:Microchemistry / Q000379:methods*; D010449:Peptide Mapping / Q000379:methods*; D013048:Specimen Handling / Q000379:methods
D007782:Lactoglobulins