Determination Of Main Antigenic Proteins In Soybean Using HPLC And HPLC-MS/MS

Soybean,with high nutrition,is one of the major vegetative protein sources used in the food and feed industries.However,soybean contains a number of antinutritional factors,which can cause adverse physiological responses,especially young animals.Of them,a subunit of soybean P-conglycinin(Gly m Bd 60K)is one major antigen protein with good thermol stability,Gly m Bd 28K is a trace protein with high antigenicity,Kunitz trypsin inhibitor(KTI)is one major antigen protein with poor thermol stability.Recent decades,many technologies have been applied to reduce the amount of antinutritional factors in soybean products.However,since variation in the processing conditions for soybean products and limitation of the detection technology are exist,there is a need to provide accurate and sensitive methods for identification and quantification of the above antigen proteins in soybean seeds and soybean products to evaluate the efficacy of the different processing methods.Our research is divided into four parts.Experiment one:quantification of Gly m Bd 60K in soybean products by high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS).Soybean protein extract was reduced with dithiothreitol,alkylated with iodoacetamide,on-filter digested by trypsin at 37℃ and analyzed by HPLC-MS/MS.NPFLFGSNR was selected as target standard peptide and the chromatographic separation was performed by XBridge(?)Peptide BEH C18(3.5 μm)with a gradient program using 0.1%formic acid in water and acetonitrile as mobile phases.Detection was performed by multiple reaction monitoring with an ESI source operated in positive ion mode.The concentration of Gly m Bd 60K was quantified in fresh samples including soybean seed,fermented soybean meal,extruded soybean meal and extruded full-fat soybean ranged from 0.0 to 41.1 mg/g with precisions(coefficient of variation,CV,%)less than 10.3%.We further analyzed the proportion of Gly m Bd 60K in P-conglycinin through the sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)analysis to determine the content of β-conglycinin in samples.The content of β-conglycinin was ranged from 0.0 to 112.4 mg/g in the above fresh soybean products.Experiment two:expression and purification of Gly m Bd 28K in E.coil.The cDNA encoding the mature Gly m Bd 28K was amplified by polymerase chain reaction(PCR).After digestion with NdeⅠ and XhoⅠ,the PCR products were ligated into the vector pET-28a.The recombinant plasmid was named as pET-28a-28K.After transformation the vertified recombinant plasmid into the E.coil BL21,single colonies were induced to yield recombinant Gly m Bd 28K using 1 mmol/L IPTG Purifiction of the recombinant Gly m Bd 28K was performed by Ni affinity chromatography.The expression level of purified recombinant Gly m Bd 28K was 5.4 g/L.The purified recombinant Gly m Bd 28K was analyzed by SDS-PAGE and electrospray ionization mass spectrometry.Results indicated that the purified recombinant Gly m Bd 28K has a molecular mass of 28 kDa and its purity was above 95%.Experiment three:quantification of Gly m Bd 28K in soybean products by HPLC-MS/MS.Soybean protein extract was treated using the same approach as the experiment one,TVVEEIFSK was selected as target standard peptide and the conditions of chromatography and mass spectrometry was performed by the same conditions as the experiment one.Results indicated that recoveries of Gly m Bd 28K in spiked soybean samples were between 99.5%and 108.1%,and intra-and interday precisions(CV,%)were less than 4.6%and 7.7%.The concentration of Gly m Bd 28K was quantified in fresh samples including soybean seed,fermented soybean meal,extruded soybean meal and extruded full-fat soybean ranged from 0.0 to 1.25 mg/g with precisions(CV,%)less than 12.5%.Experiment four:purification and quantification of Kunitz trypsin inhibitor in soybean products using offline two-dimensional liquid chromatography.The crude extract was first purified by weak anion exchange chromatography,and then the fraction containing KTI was further separated by size exclusion chromatography.The fraction containing KTI was collected and analyzed by SDS-PAGE and electrospray ionization mass spectrometry.Results indicated that purified KTI has a molecular mass of 19964.956 Da and a purity of 99%with inhibitory activity of 2425 TIU/mg protein.This assay was used for the quantification of KTI in soybean samples.The assay showed concentrations with a range between 7.8 and 500.0 pg/mL and a limit of detection of 0.12 mg/g.The recoveries of KTI in spiked soybean samples were between 82.2%and 86.7%,and intra-and interday precisions(CV,%)were less than 7.4%and 8.4%.The developed method was successfully used to analyze soybean samples from different sources and soybean products derived from different processing techniques,which demonstrated that the developed procedure provided an accurate and sensitive tool for separation and quantification of intact KTI in soybean products.