First approach based on direct ultrasonic assisted enzymatic digestion and capillary-high performance liquid chromatography for the peptide mapping of soybean proteins.
This work proposes, for the first time, the use of a high intensity ultrasonic probe to accelerate the tryptic digestion of soybean proteins. Different digestion parameters were optimized: protein extracting solution, reduction, and alkylation conditions (time, concentration, and temperature), trypsin:protein ratio, and ultrasonic conditions (sonication amplitude and time). Separation of peptide profiles was carried out by capillary-HPLC. The effect of the variation of chromatographic conditions (elution gradient, column temperature, and injection volume) on peptide separation was also studied using two capillary-HPLC columns with different column diameters and particle sizes. Moreover, samples were focused at the top of the column in order to obtain an increasing sensitivity without loss of efficiency. This method was successfully applied to the profiling of soybean peptides from transgenic and non-transgenic soybeans and from different pigmented beans commercialized as soybeans.
Domínguez-Vega
Elena
E
Department of Analytical Chemistry, Faculty of Chemistry, University of Alcalá, Ctra Madrid-Barcelona Km 33.600, 28871 Alcalá de Henares, Madrid, Spain.
García
Ma Concepción
MC
Crego
Antonio L
AL
Marina
María Luisa
ML
D002851:Chromatography, High Pressure Liquid; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D010449:Peptide Mapping / Q000379:methods*; D030821:Plants, Genetically Modified; D030262:Soybean Proteins / Q000032:analysis / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism*; D014465:Ultrasonics
D010446:Peptide Fragments; D030262:Soybean Proteins; D014357:Trypsin