Detection Technology Of Honey Authenticity By Liquid Chromatography-mass Spectrometry Based On Proteomics

Adulteration is a long-standing problem in the honey industry and has resulted in very serious impact on the honey industry.The current detection method was useful in the detection of known honey adulterants,but it lacks the ability of early warning and rapid response for new honey adulterant,and has the disadvantages that the detection technology development always lags behind the adulteration technology and costly.Thus,it is difficult to eliminate the honey adulteration phenomenon.In recent years,biomarker detection technology based on proteomics and metabolomics is widely used in the field of food authenticity detection,It has laid a foundation for the study of using major royal jelly proteins(MRJPs)as a biomarker for honey authenticity detection.However,due to the low protein content in honey,there was no useful methods for MRJPs quantitative detection.In this paper,a quantitative analysis method for MRJP1～3 in honey was establishes by liquid chromatography tandem mass spectrometry detection technology.The MRJP 1～3 content database in natural honey was established using a large number of natural honey samples,and the honey authenticity detection threshold was set.The effectiveness of this method in honey authenticity test is verified by the existing honey adulteration detection method.1.Qualitative identification of MRJPsThe MRJPs in royal jelly were qualitative identification by ultra performance liquid chromatography tandem time-of-flight mass spectrometry(UPLC-Q-TOF)after tryptic digested.Protein Lynx Global Server(PLGS)software was used to analyze the obtained peptide sequence data from UPLC-Q-TOF based on the theoretical information of MRJPs obtained from the Uni Prot database.The primary sequence data of 8 kinds of royal jelly proteins(MRJP 1～7 and MRJP 9)were successfully identified.There are total 108 peptides,24 belong to MRJP1,28 belongs to MRJP2,21 belongs to MRJP3,14 belongs to MRJP4,11 belongs to MRJP5,3 belongs to MRJP6,e 6 belongs to MRJP7 and only 1 belongs to MRJP9.The peptides coverage of 8 major royal jelly proteins is ranged from 1.7%～76.6%.2.Selection of candidate marker peptides for MRJPs in honeyFirst,the reproducibility of all the candidate peptides of royal jelly protein in honey was analyzed by ultra performance liquid chromatography tandem triple quadruple mass spectrometry(UPLC-TQMS).34 peptides with high peak area and good reproducibility were initially selected from 69 candidate polypeptides(the only peptide of MRJP 9 could not be detected because of its low response value,so it was removed).All the candidate peptides were selected according to the two criteria based on digestibility and stability.All of candidate peptides were divided into five categories:(1)A,high digestibility and stability;B,high digestibility but poor stability;C,high stability but poor digestibility;D,poor digestibility and stability;E,unknown.One specific marker peptide YNGVPSSLNVISK belonging to class B was successfully screened for MRJP 1;One specific marker peptide TLQMIAGMK belonging to class A was screened for MRJP 2;One specific marker peptide LTVAGESFTVK belonging to class A was screened for MRJP 3.3.Establishment and validation of quantitative method for MRJP 1～3 in honeyAfter synthesizing three specific marker peptide standards and their isotope-labeled peptides for MRJP 1～3 separately,an ultra-high performance liquid chromatography tandem triple quadrupole mass spectrometry(UPLC-TQMS)method was established for the quantitative detection of MRJP 1～3 in honey.Method validation of this method was carried out by standard curve,precision,recovery and limit of quantitation analysis,the results show that this method is scientific and reasonable,Meet the testing requirements.Four common honey(Acacia honey,rape honey,litchi honey and chaste honey)and three common syrup adulterations(corn syrup,rice syrup and high fructose syrup)were quantitative analyzed by UPLC-TQMS.The results showed that 3 specific marker peptides of MRJP 1～3 can be detected in all the honey samples,but none can be detected in corn syrup,rice syrup or high fructose syrup,which confirmed that MRJP 1～3 are specific endogenous protein in honey.The results of simulated syrup adulteration experiment showed that the concentration of the added high fructose syrup is inversely related to the content of MRJP 1～3 in honey sample(R ~2>0.99),confirmed that this method could be used for detection of partial adulterated honey.4.Establishment and application of MRJP 1～3 content database in real honeyThe MRJP 1～3 content of 70 authentic honey samples were quantitative analyzed by UPLC-TQMS,and the MRJP 1～3 content database containing acacia honey,rape honey,litchi honey and chaste honey was established.The correlation analysis of the MRJP1～3 content database showed that there is a significant difference in the total content of MRJP 1～3 between different honey categories,confirmed that this method can be used for the identification of honey category.Different real thresholds of MRJP 1～3 content was set based on the real content database of MRJP 1～3 for four honey categories,respectively.The effectiveness of this honey authenticity test method by UPLC-TQMS was confirmed by the similar identification result of 9 commercially honey after compared with stable carbon isotope ratio mass spectrometry(IRMS)method from national standard method.