Comparative Study And Application Of Methods For Determination Of Peptide Concentration

Objective With the development of proteomics and peptide omics,rapid acquisition of peptide concentration is important for studying the properties and mechanism of peptides.At present,the method for determining the concentration of peptides in the laboratory has its own advantages in terms of difficulty,scope of application,economy.Therefore,we need to propose a rapid and simple method to determine the peptide concentration.Method Bovine serum albumin?BSA?and solid phase synthesis of cyclic peptide CYE?NH₂-CYENLRSTC-COOH,C₁-C₉disulfidebondinto ring?,CLP?NH₂-CLPLWYPSC-COOH,C₁-C₉ disulfide bond into ring?and the CHY?NH₂-CHYPTYSKC-COOH,C₁-C₉ disulfide bond into ring?solutions were serially diluted.The extinction coefficient method and the BCA method were used to measure the absorbance of the solutions.The corresponding concentration values were calculated by the extinction coefficient formula or the standard curve equation.The differences between the calculated value and the theoretical value were compared,and the precisions of the measurement methods were calculated.The peptides CYE and 1018?VRLIVAVRIWRR?solutions which may have different affinities on hydroxyapatite powder surface were serially diluted,and the BSA was used as a control.CYE,1018 and BSA were incubated with 25 mg/mL hydroxyapatite suspension at 37?.After 1 hour,the supernatant was taken after centrifugation and the concentration was determined by BCA method using a personalized standard curve.The adsorption amount of BSA,CYE and 1018 on the surface of hydroxyapatite powder was evaluated according to the concentration calculation results.Result There was no significant statistical difference between the concentration calculation value obtained by extinction coefficient method and the theoretical value?P>0.05?.In the BCA method,there was a statistically significant difference between the theoretical value and the calculated value of the peptide concentration using BSA as a standard?P<0.05?.There was no statistically significant difference between the calculated and theoretical values of the peptide concentration obtained from the peptide itself as a standard?P>0.05?.Plotting the individualized standard curve by the peptides which had the approximate length and molecular weight with the peptide to be tested,there was a statistical difference between the calculated value and the theoretical value?P<0.05?.However,the calculated concentration is closer to the theoretical value than the calculated value of the BSA standard curve?P<0.05?.There was no significant difference in precision between the methods?P>0.05?.The adsorption capacity of BSA,CYE and 1018 on hydroxyapatite powders was evaluated by the BCA method using the personalized standard curve drawn by the peptide itself.The results showed that the adsorption capacity of CYE and 1018 reached the plateau when the concentration reached 75 g/mL,while the BSA showed an increasing trend.Conclusion The extinction coefficient method can be used when the polypeptide contains tryptophan or tyrosine.When the property of the standard substance is closer to the peptide be tested,the more accurate the concentration calculated by BCA method using personalized standard curve is,which can be used for rapid semi-quantitative determination of peptide concentration.