| Tuna is a globally recognized high-quality marine fish species with high protein and low fat.The facility aquaculture technology of tuna has not been popularized yet due to the unique large-scale migratory survival characteristics of tuna.The severe depletion of wild resources has led to high prices for tuna.High-quality tuna is unavailable in fish market sometimes.There are significant differences in nutritional value among different tuna species,as well as their commercial prices accordingly.Therefore,the risk of adulteration driven by economic benefits is extremely high.In addition,different species are named as “Tuna” worldwidely,which can easily lead to ambiguity and hinder market transparency,thus causing harm to the management of tuna.To prevent the fraudulent behavior of merchants,maintain the market order,and protect the rights and interests of consumers,it is particularly important to identify different tuna species.Currently,commercialized tuna products are usually sold after processing,which leads to the elimination of morphological features and certain degradation of DNA during the processing process either lightly or deeply processed.Therefore,traditional morphological and molecular biology methods have inevitable limitations in practical applications.Conventional physical and chemical analysis techniques are insufficient in sensitivity,specificity,robustness,etc,and they cannot provide enough information for reflecting the internal molecular composition of tuna,which also had severe limitations in practical application.Varying with species,tuna contains unique nutritional and functional molecules such as proteins,lipids and metabolites.The omics analysis technology based on chromatography-mass spectrometry has been successfully applied in meat products,aquatic products,dairy products,honey,vegetable oils,and other products,providing important inspiration for exploring the intrinsic relationship between the nutritional functional components of tuna and tuna species.As a breakthrough to the limitations of existing analytical techniques in tuna species identification,multiple-omics analysis based on chromatography-mass spectrometry was adopted in this study,from the multidimensional perspective of proteomics,lipidomics and metabonomics,to analyze the characteristic substances of three different tuna species including skipjack tuna,bigeye tuna and yellowfin tuna.Chemometric and bioinformatic tools were used for analyzing the omics data and clarifying differential biomolecules of three tuna species.Thus,a multi-omics analysis system for species differences was established.Usually,the workload of species identification is large and the program is complex and timeconsuming.The identification of characteristic substances is of great significance for tuna species identification.Additionally,the multi-omics analysis system established in this study also provides technical support for nutritional research and bioactive substance discovery for tuna.The main conclusions of this study are as follows:(1)Differential analysis and species-specific peptide identification of three tuna speciesThree tuna species including skipjack tuna,bigeye tuna and yellowfin tuna were used as the research object in this study.The tryptic-digested peptides were separated and detected by ultrahigh-performance liquid chromatography-quadrupole-time of flight mass spectrometry(UPLC-Q-TOF/MS)using data-dependent acquisition(DDA)mode.The peptides were identified by Protein Pilot software.The differential peptides in the three different tuna species were screened by Venn analysis.And the species specificity of the differential peptides was analyzed by the bioinformatic tool National Center for Biotechnology Information’s Basic Local Alignment Search Tool(NCBI BLAST).Finally,the detection specificity of species-specific peptides was verified by multiple reaction monitoring(MRM)mode based on a triple quadrupole mass spectrometer.Through MRM validation,three,one and two species-specific peptides were confirmed from skipjack tuna,bigeye tuna and yellowfin tuna respectively,which were mainly from myoglobin,myosin light chain and cytochrome c oxidase.A rapid detection method based on MRM mode on a triple quadrupole mass spectrometry was established in this study.(2)Identification of differential proteins of three tuna species by SWATH-MSbased proteomics These differential lipids all belong to the glycerol phospholipids,including phosphatidyl choline(PC),lysophosphatidyl choline(LPC),phosphatidyl ethanolamine(PE),lysophosphatidyl ethanolamine(LPE),phosphatidyl glycerol(PG),and phosphatidyl inositol(PI).Hierarchical cluster analysis was carried out on three tuna samples through differential lipids.The results showed that three tuna species formed their own independent clusters,and the unmodeled samples were correctly clustered into their respective classifications,indicating that the screened differential lipids had the potential ability for tuna species differentiation.Based on the spectrum library established through data-dependent acquisition,the protein relative quantitation of three tuna species was carried out using sequential window acquisition of all theoretical fragment ions(SWATH)data-independent acquisition(DIA)mode.The relative quantitative data of proteins were obtained through SWATH data collection,and ultimately 81 proteins were reliably quantified.Chemometric models for tuna species differentiation were constructed based on protein relative quantitative data,including the principal component analysis(PCA)model and the orthogonal partial least squares-discriminant analysis(OPLS-DA)model.Three tuna samples were clearly distinguished in the principal component analysis.Differential proteins of three tuna species were screened through pairwise comparisons of OPLS-DA models.A total of 14 differential proteins were screened through three pairwise comparisons of OPLS-DA models,including myosin,myoglobin,hemoglobin,parvalbumin,titin-like protein,and calcium ion transporting ATPase.Hierarchical clustering analysis was carried out on three tuna samples through the screened differential proteins.The results showed that three tuna samples formed their own independent clusters,indicating that the screened differential proteins had the potential ability for tuna species differentiation.Finally,the bioinformatics tool Uniport was used to annotate the biological functions of differential proteins.(3)Identification of differential lipids of three tuna species through lipidomics based on high-resolution mass spectrometry and chemometricsThe lipid profiles of three tuna species were characterized by ultrahighperformance liquid chromatography-quadrupole orbitrap mass spectrometry(UPLCQ/Orbitrap/MS)through data-dependent acquisition in both positive ion mode and negative ion mode.A total of 212 and 227 lipid molecules were identified and relatively quantified in the positive ion mode and negative ion mode respectively through MSDIAL software.OPLS-DA models among different tuna species were constructed using relative quantitative data of lipids.Differential lipids of three tuna species were screened through pairwise comparisons of OPLS-DA models.As a result,a total of 27 differential lipids were screened from three pairwise comparisons of OPLS-DA models.(4)Identification of differential metabolites of three tuna species through metabonomics based on high-resolution mass spectrometry and chemometricsMetabonomics based on high-resolution mass spectrometry was used to detecte the metabolites of three tuna species through gas-chromatography-quadrupole/time of flight mass spectrometry(GC-Q-TOF/MS)in positive ion mode and UPLCQ/Orbitrap/MS in both positive ion mode and negative ion mode.The metabolites in three tuna species were identified and relatively quantified by MS-DIAL software.A total of 22 metabolites were identified by GC-Q-TOF/MS-based metabonomics.And38 and 44 metabolites were identified by UPLC-Q/Orbitrap/MS-based metabonomics in the positive ion mode and negative ion mode respectively.These metabolites were mostly amino acids and their derivatives,organic acids,and some nucleic acid components,fatty acids,sterols,amines,etc.were also identified.Chemometrics models for tuna species differentiation were constructed through metabonomic data from UPLC-Q/Orbitrap/MS.A total of 38 differential metabolites were screened from three pairwise comparisons of OPLS-DA models.Hierarchical cluster analysis was carried out on three tuna samples through differential metabolites.The results showed that three tuna species formed their own independent clusters,indicating that the screened differential metabolites had the potential ability for tuna species differentiation.The Kyoto Encyclopedia of Genes and Genomes(KEGG)was used to analyze the pathways involved in these differential metabolites.The results showed that a total of27 metabolic pathways were annotated,including D-glutamine and D-glutamate metabolism,phenylalanine,tyrosine and tryptophan biosynthesis,phenylalanine metabolism and arachidonic acid metabolism,etc.Finally,the correlation analysis of the screened differential metabolites with differential proteins and differential lipids was performed using a correlation matrix heatmap and correlation network diagram visually. |
PDF Full Text Request