Determination Of A1 And A2 ?-casein In Milk By Liquid Chromatography-high Resolution Tandem Mass Spectrometry

Milk and its products are one of the most important foods in daily life.Its rich protein can provide nutrition for people of all ages.Casein is the protein with the highest proportion in milk and mainly divided into ?,? and ?-casein,of which ?-casein accounts for about 40%.?-casein in milk is mainly classified as A1 and A2 ?-casein,in which one amino acid residue differs at 67 th position.This difference causes the two proteins to produce different bioactive peptides after being human digested.Intake only A2 ?-casein could reduce the risk for heart disease and diabetes,etc.Therefore,it is of great importance to establish a method determining A1 and A2 ?-casein for development of A2 dairy product,and quality inspection of A2 dairy product.Due to the difficulty and high cost of detecting intact protein,this study established a new method based on liquid chromatography-high resolution tandem mass spectrometry(LC-HRMS/MS)to detect the digested products of casein.Caseins were extracted from milk based on isoelectric precipitation,followed by trypsin and thermolysin digestion.Digested casein products were subjected to LC-HRMS/MS analysis.The peptide containing varied amino acid moiety at the 67 th position was selected as characteristic species to specifically distinguish A1 and A2 ?-casein.The main work is as follows:1.During the sample preparation process,tris-HCl aqueous solution was used to extract caseins,which avoids desalting procedure.The addition of acetonitrile,used as a protein denaturation reagent,significantly improves the digestion efficiency.According to the actual measurement,the best condition appeared when the acetonitrile amounts for 25%(v/v),so that the digestion of casein can be completed within 6 hours.During the thermolysin experiment,the digestion system was further simplified,and nothing except tris-HCl was added.The casein digestion was promoted by increasing the digestion temperature to 60?,so that the reaction can be completed within 4 hours.2.The characteristic peptides generated in the trypsin experiment were same in length and similarity in composition,while the characteristic peptides in the thermolysin experiment were quite different.Two sets of liquid chromatography gradient elution programs were developed and optimized.After optimizing all the mass spectrometry parameters using the synthetic peptide standards Simultaneous adoption of high resolution mass spectrometry and tandem mass spectrometry(under CID mode)avoids the false signal possibly generated from impurity,which increases the reliability of results.3.4 normal milk samples and 2 A2 milk samples were tested using these two methods,the liquid chromatography retention time,mass spectrum and CID data were highly consistent with theoretical data.The results showed that the content of A1 ?-casein was about 2-4 times higher than A2 ?-casein in a normal milk,while a small amount of A1 ?-casein existed in one A2 milk sample.In summary,the method detecting characteristic peptides constructed in this paper can be used to determine A1 and A2 ?-casein in milk.The specificity,detection efficiency and reliability can be greatly improved by using two different enzymes,a new digestion system and an LC-HRMS/MS equipment.In addition,detecting only the fragments of casein can reduce the requirements for detection equipment,which made this method better for A2 raw milk source screening and quality control.