Study On Meat Protein N-glycome Analysis And N-glycan Profile As Biomarker In Common Meat Adulteration Detection

Meat is one of the most popular foods of consumers due to it high nutritious value and unique flavor.Driven by the high profits,the problem of adulteration of meat and meat products occurs frequently in meat industry and market.Mass spectrometry,PCR,infrared spectroscopy,and hyperspectral techniques are currently common methods for adulteration detection of meat and meat processed products,however,each of them has its own drawback.It is thus necessary to discover novel robust and sensitive methods engaging both qualitative and quantitative analysis for meat authentication detection to minimize the drawbacks of the existing methods and meet the needs of market monitoring.Protein N-glycosylation is one of the most important post-translational modification of proteins.Different N-glycans structures are formed in this period and reflect the genetic background of the cell during the expression process which makes N-glycans specific among species.Therefore,it is of great importance to develop a new meat adulteration detection method based on N-glycan structure difference.In this study,beef,chicken,pork,duck meat and mutton were used as research objects.Meat N-glycans extraction method was established and UPLC was used to separate and detect N-glycans,the structure analysis was carried by MALDI-TOF-MS/MS.To explore the role of N-glycans played in meat adulteration detection,two methods were established.One was to identify binary adulterated meat samples,trinary adulterated meat samples and processed adulterated meat samples based on meat N-glycan UPLC profile analysis.The other was distinguishing pork and duck meat adulterated beef samples based on species-specific N-glycans UPLC-ESI-MS/MS detection.The specific research contents are as follows:1.Extraction,UPLC analysis and structural composition analysis of N-glycomes derived from five meat speciesThree N-glycans enzyme digestion pre-treatment methods were designed by taking beef as model,including three meat protein preparation methods(homogenate extraction,ultrasonic extraction and direct digestion).And the direct digestion method was chosen for other meat species N-glycans extraction.For six repeats,the RSD values of each UPLC peaks were under 10%which exhibited good reproducibility of the established N-glycans extraction method.Then,using this method to extract N-glycomes from beef,chicken,pork,duck meat and mutton.UPLC was used to obtain the single N-glycan peaks,which were further detected by MALDI-TOF-MS/MS.N-glycans structures were deduced by MS date and GU value.The results showed that the structures of N-glycans in five meat species were significantly different.Among them,19,22,20,19,and 22 N-glycans structures were detected in beef,chicken,pork,duck,and mutton,respectively.The composition of N-glycan in the five meat species was obviously different based on the analysis of the proportions of the three main N-glycan types,including high mannose,fucosylation and sialylation.2.Meat adulteration detection based on N-glycan UPLC profile analysisN-glycan peaks with a relative peak area percentage above 1%in five meat UPLC profiles were obtained to build dataset and analyzed by PCA.The result showed beef,chicken,pork,duck meat and mutton can be separated from each other based on N-glycan profile analysis.To further verify the applicability of this N-glycosylation based method in meat authentication,beef spiked duck meat and beef spiked both pork and chicken were examined.PCA and PLS were performed to obtain qualitative and quantitative results.The result showed that the established method can successfully applied in qualitative and quantitative analysis of binary species and trinary species meat adulteration detection,and the LOD was found to be 2.2%.Furthermore,the N-glycan profiles of meats were shown to stay stable upon various processing procedures such as boiling,roasting,frying,microwave and high-pressure treatments,indicating that our method can be employed in processed meat adulteration identification.3.Meat adulteration detection based on characteristic N-glycan structure UPLC-ESI-MS/MS analysisBeef characteristic N-glycan F1M5A1G1S1(Ac),pork characteristic N-glycan M5A2G1G2(?)S1(Ac),duck meat characteristic N-glycans F1A3G1?A3G2?F1A3G2 were detected by UPLC-ESI-MS and their theoretical m/z in MS were 1118.90[M-2H]2-?1207.65[M-2H]2-?914.85[M+2H]2+?922.85[M+2H]2+,and 995.85[M+2H]2+,respectively.The MRM qualitative and quantitative identification of duck meat spiked beef or pork spiked was established based on the parent ions and three high response secondary mass spectrometry product ions of each characteristic N-glycans structures.The result showed the established method can be efficiently applied in discriminating adulterated meat samples from pure beef sample.Besides,the LOD of pork and duck meat were found to be 0.88%and 0.26%,average recovery rate of this method was between 105%-115%.