LC-MS/MS-based Quantification Of Proteins At Adsorption Layer Of Emulsion Stabilized By Legume Proteins

Proteins are one of important emulsifiers in food products.Many food proteins are amphiphilic molecules capable of adsorbing to the surface of oil droplets and forming viscoelastic films,thus stabilizing the droplets against flocculation and coalescence.The formation and stability of emulsion stabilized by protein are closely related to the interfacial properties of protein.To investigate the adsorbed proteins,most previous studies focused on the structure,physicochemical characteristics and rheological properties of proteins at adsorption layer.Most research on the quantitative information has been limited to the comparison between interfacial protein and protein in the continuous phase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE）,therfore with mere semi-quantitative results obtained.Legume protein isolates are a mixture of various proteins.Little literature was available on the interfacial behaviors of legume protein isolates due to the complexity of these proteins.This work adopted the method of quantitative proteomics for the identification and quantification of proteins at adsorption layer of pea protein isolate（PPI）-stabilized emulsion.Then,the adsorption and dilatational rheology of pea proteins（PPI,albumin（2S）,vicilin（7S）and legumin（11S））at the oil-water interface were monitored,and the correlation analysis was combined with the mass spectral results.Moreover,soy protein isolate（SPI）was subjected to the established analytical approach for further verification of the applicability,and the effect of heat treatment on adsorbed protein was determined.The main research contents and results were indicated as follows:1.This study adopted the method of quantitative proteomics to analyze the adsorbed proteins in oil-in-water emulsions stabilized by PPI.Adsorbed proteins were precipitated by an optimized precipitation method and precipitates were labeled and subjected to a reversed-phase high performance liquid chromatography coupled to tandem mass spectrometry（RPLC-ESI-MS/MS）for protein identification and quantification.In total,77 proteins were identified,of which 49 proteins with significant differences were observed.There were 25 upregulated proteins（fold change>1）and 24 downregulated proteins（fold change<1）.The interfacial adsorption abilities of these proteins were compared according to the classification of protein families.The results showed that all isoforms of vicilins exhibited high adsorption abilities at the oil-water interface.Compared with vicilin,convicilin showed opposite adsorption capacity.Different legumin families showed significantly different affinities on the oil-water interface.In contrast to albumin-1,albumin-2 was preferentially adsorbed to the interface.The amino acid sequence alignment and hydropathy profile analysis of these proteins showed that the proteins well-balanced between hydrophobic and hydrophilic amino acid groups displayed high interfacial activity.In contrast,a long hydrophilic or hydrophobic fragment could adversely influence protein interfacial activity.2.In this study,the differences in the structure and adsorption and dilatational rheology at the oil-water interface of heat-treated pea proteins（PPI,11S,7S,2S）were compared.It was found that the protein type and the flexible structure of protein influenced on the interfacial property by determining the related structural properties,including protein unfolding,particle size,zeta potential,surface hydrophobicity and free thiol content.Compared with 11S,PPI and 7S,2S had larger particle size,lower solubility and lower surface hydrophobicity,thus the smallest apparent diffusion rate(K_diff)and surface pressure at the end of adsorption(7200 s,π₇₂₀₀)were obtained.Compared with native pea proteins,heat treatment（95℃,20 min）resulted in protein unfolding,and surface hydrophobicity and sulfhydryl content changes.Therefore,the heat-treated protein（c=0.05%）showed higher surface activity,and the diffusion rate followed the trend of PPI>11S>7S>2S,of which 11S was equal to 7S.In addition,each protein formed a viscoelastic film at the interface and primarily elastic in nature.PPI established the largest intermolecular interactions at the interface,followed by 11S,7S and 2S.The intermolecular interaction of 2S was significantly enhanced by heat treatment or the increase of the concentration.Compared with quantitative study results,the poorer adsorption capacity of 2S was verified.3.Quantitative proteomics was adopted to identify and quantitate the adsorbed proteins in oil-in-water emulsions stabilized by native or heated SPI.Adsorbed proteins were analysed by RPLC-ESI-MS/MS.In total,95 proteins were identified,of which 54proteins with significant differences were observed between adsorbed native proteins and protein in the continuous phase.There were 27 upregulated proteins（fold change>1）and27 downregulated proteins（fold change<1）.And there were 50 proteins with significant differences were observed between adsorbed native proteins and adsorbed heated proteins,of which 28 upregulated proteins（fold change>1）and 22 downregulated proteins（fold change<1）were obtained.The results showed that heat-treated（95°C,20 min）protein exhibited a larger K_diff and a shorter equilibrium adsorption time due to protein unfolding and more flexible conformation.Albumin（2S）and glycinin（11S）exhibited low adsorption abilities at the oil-water interface.Compared withβ-conglycinin of 7S family,Basic 7S globulin showed opposite adsorption capacity.Basic 7S globulin was preferentially adsorbed to the interface.This might be attributed to the balance between hydrophobic and hydrophilic amino acid groups.In addition,isotope labeling combined with LC-MS/MS techniques further demonstrated that the interfacial behavior of protein was influenced by heat treatment,and the preferential adsorption of globulin and part of albumin in heated SPI was obtained.This study provides a detailed quantification information of native or heated protein at interfacial layer and it lays the foundations for better understanding of the interfacial behaviors of proteins.