Identification And Quantitative Analysis Of Characteristic Proteins And Peptides Of Edible Bird’s Nest Based On LC-MS

Edible bird’s nest(EBN)has a long history of application in China.The main component of EBN is protein,and studies have shown that it has various effects such as anti-viral,antioxidant,whitening and moisturizing.However,the existing method of EBN identification based on the total protein content has room for improvement.Therefore,it is important to develop a reliable method for the analysis and identification of edible bird’s nests.In this work,we established the visualization of EBN characteristic protein gel map and identified the specific proteins in it.A protein and peptide analysis library containing information on raw materials,finished products and adulterants of EBN was established by using in-depth proteomics technology.The above information was combined to identify the characteristic peptides of EBN and a method for quantitative analysis of the characteristic peptides was developed based on UPLC-QTOF-MS.Finally,the method was applied to the authentication of raw materials and commercially available edible bird’s nest products.The main findings are as follows:(1)Gel mapping of EBN proteins and identification of their characteristic proteinsRaw materials of EBN from different origins,different brands of commercially available stewed products,and EBN-related adulterants were collected.The extraction methods of EBN proteins were screened,and the samples were subjected to SDS-PAGE gel electrophoresis to establish a visualized gel profile of the characteristic proteins.The protein bands on the gels were subjected to in-gel trypsin digestion and proteomic analysis to identify the proteins and peptides in the different bands.Seven to 18 proteins and 11 to 33 peptides were identified in eight bands with low to high molecular weights,distributed in the proteomes of three species of Chaetura pelagica,Hemiprocne comata,and Hirundo rustica rustica,respectively.These proteins include Acidic mammalian chitinase,Lysyl oxidase homolog,MUC5AC,already reported in EBN,and MGLL lipase,RRP5 protein,found in EBN for the first time.(2)Establishment of protein and peptide libraries for EBN identification and screening of characteristic peptidesRaw EBN from different origins,finished commercial products from different brands and related adulterants were subjected to protein extraction and mixing,and trypsin digestion and proteomics analysis.A total of 1167 proteins and 5435 peptides were obtained from three parallel experiments.Sixty-five,232,and 80 peptides and 17,53,and 34 proteins were identified from the proteomes of three swallow species,including Chaetura pelagica,Hemiprocne comata,and Hirundo rustica rustica,respectively.Another 1 to 524 proteins were identified in the proteomes of 21adulterant-related species,including soybean,chicken,and silver fungus.In order to screen the peptides for quantitative analysis,the peptides identified in different molecular weight bands were compared with the established EBN identification protein and peptide library,and 21 peptides were obtained,which were distributed in four different EBN proteins.Further screening of their length and number of post-translational modifications yielded characteristic peptides for subsequent quantitative analysis,namely EMVAAFEQEAR,LLVGFPTYGR,YPLITTLK,FSSQIHNNGR,LANWPYGHR,LEGAVIELTR,and SLSEPDVTCSTK.(3)Establishment of an edible bird’s nest identification method based on quantitative analysis of characteristic peptidesThe separation of seven candidate peptides was established in a UPLC-QTOF-MS system,and the five peptides with high peak areas(EMVAAFEQEAR,LLVGFPTYGR,YPLITTLK,LANWPYGHR,LEGAVIELTR)were selected for subsequent methodological validation after the pre-analysis of the raw EBN samples.Linearity analysis showed good linearity for the five peptides with R~2 of 0.995,0.9954,0.9997,0.9972 and 0.999,respectively.The limits of detection(LOD)were 0.05,0.004,0.0003,0.05,and 0.01μg/m L,respectively.the limits of quantification(LOQ)were 0.15,0.13,0.01,0.15,and 0.04μg/m L,respectively.and the intra-day and inter-day precision and recoveries were validated.In addition,the contents of these five peptides were below the detection limits in the adulterated samples of pig skin,white fungus,gelatin,swim bladder,egg white and soy protein,indicating that they could be used for the analytical identification of edible bird’s nests.(4)Quantitative analysis of characteristic peptides in raw materials and products of edible bird’s nestsThe quantitative analysis of characteristic peptides was performed for 16 EBN raw materials from different origins and different suppliers,such as Malaysia,Thailand and Indonesia,and 6 different brands of commercially available products commonly available in local market.The average contents of the 5 peptides in raw materials were0.573,1.032,0.323,0.390 and 0.234μg per mg of protein,respectively.Among the six brands of commercial products,the total concentrations of characteristic peptides were10.6,11.2,3.8,10.1,13.3,and 10.1μg/m L,respectively.The method of tracing the origin of EBN as well as the quality of stew products in market was further discussed by statistical analysis of the UPLC chromatogram and the content of the characteristic peptides.The novelties of this work are:(1)The first edible bird’s nest protein gel profile was established and several first proteins were identified from different bands;the first edible bird’s nest identification protein peptide library was established,which includes proteins and peptides that may occur in edible bird’s nest and its adulterants,providing an important reference for the subsequent research.(2)A quantitative analysis method was developed based on the identification and screening of the characteristic peptides of EBN,which provides an important tool for quality control and authenticity identification.(3)The content of characteristic peptides in raw materials from different sources was analyzed and statistical analysis was performed to discuss the relationship between characteristic peptides and sources.The contents of characteristic peptides in different brands of commercially available stewed products were analyzed and their quality was evaluated.