Pheretima,also called“earthworms”,is a well-known animal-derived traditional Chinese medicine that is extensively used in over 50 Chinese patent medicines(CPMs)in Chinese Pharmacopoeia(2020 edition).However,its zool...Pheretima,also called“earthworms”,is a well-known animal-derived traditional Chinese medicine that is extensively used in over 50 Chinese patent medicines(CPMs)in Chinese Pharmacopoeia(2020 edition).However,its zoological origin is unclear,both in the herbal market and CPMs.In this study,a strategy for integrating in-house annotated protein databases constructed from close evolutionary relationship-sourced RNA sequencing data from public archival resources and various sequencing algorithms(restricted search,open search,and de novo)was developed to characterize the phenotype of natural peptides of three major commercial species of Pheretima,including Pheretima aspergillum(PA),Pheretima vulgaris(PV),and Metaphire magna(MM).We identified 10,477 natural peptides in the PA,7,451 in PV,and 5,896 in MM samples.Five specific signature peptides were screened and then validated using synthetic peptides;these demonstrated robust specificity for the authentication of PA,PV,and MM.Finally,all marker peptides were successfully applied to identify the zoological origins of Brain Heart capsules and Xiaohuoluo pills,revealing the inconsistent Pheretima species used in these CPMs.In conclusion,our integrated strategy could be used for the in-depth characterization of natural peptides of other animal-derived traditional Chinese medicines,especially non-model species with poorly annotated protein databases.展开更多
Peptidomics draws more and more attention in discovering useful biomarkers for early diagnosis of disease. However, there is lack of efficient quantification strategy in peptidome analysis. In this study, a strategy w...Peptidomics draws more and more attention in discovering useful biomarkers for early diagnosis of disease. However, there is lack of efficient quantification strategy in peptidome analysis. In this study, a strategy with label-free quantification of the targeted endogenous peptides based on peak intensity using μUPLC-Q-TOF-MS/MS was developed for quantitative peptidome analysis of human serum. Different amounts of standard BSA tryptic digesting peptides were added into the same serum extracts for evaluation of the developed strategy, and it was observed that the average relative error of the targeted peptides was 6.42%, which was superior to the result obtained directly by commercially available software PLGS. It was also demonstrated that this quantification strategy could obviously increase the detection sensitivity of the peptide by DDA analysis. Then, this strategy was applied to comparatively analyze the peptides extracted from the serum of HCC or breast cancer patients and healthy individuals, respectively. Peptides with charge states up to 5 and molecular weight over 4000 can be reliably identified and quantified. This quantitative analysis method based on μUPLC-Q-TOF-MS/MS exhibited superior sensitivity than that by MALDI-TOF-MS commonly used in peptidome analysis. Finally, some interesting endogenous peptides related to corresponding diseases were successfully obtained.展开更多
Pyroglutamyl(pGlu)peptides are formed from intramolecular cyclization of glutamine or glutamic acid residue at the N-terminal position of peptides.This process can occur endogenously or during processing of foods cont...Pyroglutamyl(pGlu)peptides are formed from intramolecular cyclization of glutamine or glutamic acid residue at the N-terminal position of peptides.This process can occur endogenously or during processing of foods containing the peptides.Some factors such as heat,high pressure and enzymatic modifications contribute to pGlu formation.pGlu peptides are thought to have different characteristics,especially bitter and umani tastes,and thus can affect the sensory properties of foods that contain them.Moreover,some health-promoting properties have been reported for pGlu peptides,including hepatoprotective,antidepressant and anti-inflammatory activities.However,the role of pGlu residue in the peptide bioactivity is not completely established,although the hydrophobic-lactam ring is thought to enhance the peptide stability against degradation by gastrointestinal proteases.This review discusses the occurrence and formation of pGlu peptides in foods,their quantification,sensory and biological properties,and prospects in food applications.展开更多
Studying the genetic basis and regulatory mechanism of chrysanthemum lateral bud outgrowth is of great significance for reduction the production cost of cut chrysanthemum.To clarify the molecular basis of lateral bud ...Studying the genetic basis and regulatory mechanism of chrysanthemum lateral bud outgrowth is of great significance for reduction the production cost of cut chrysanthemum.To clarify the molecular basis of lateral bud elongation after removal of apical dominance in chrysanthemum,label-free quantification analysis was used to analyze the proteome changes after apical bud removal.Quantitative real-time PCR(qPCR)was used to analyze the changes in the expression of three plant hormone-related genes.A total of 440 differentially expressed proteins were successfully identified at three time points during the lateral bud elongation.The number of differentially expressed proteins in the three stages(24 h/0 h,48 h/0 h,48 h/24 h)were 219,332,and 97,respectively.The difference in expressed proteins in the three comparison stages mainly involves RNA processing and modification;translation,ribosomal structure and biogenesis;Posttranslational modification,protein turnover,and chaperones.Path analysis showed that there was various physiological activities in the process of lateral bud dormancy breaking and elongation,which involved energy metabolism,biosynthesis,signal transduction and stress response in the growth process of lateral buds.qPCR indicated that the expression of cytokinin synthesis related gene was significantly increased after the removal of apical dominance,while the expression of strigolactones synthesis related gene experiences a dramatic fall to promote the development of the lateral buds.However,there was a drop before a slight increase in the expression of the auxin synthesis related gene,which was mainly due to the removal of apical dominance that led to the loss of indoleacetic acid in the main stem.However,with formation of the new apical source,indoleacetic acid can be released again.展开更多
Label-free quantification is a valuable tool for the analysis of differentially expressed proteins identified by mass spectrometry methods.Herein,we used a new strategy:data-dependent acquisition mode identification c...Label-free quantification is a valuable tool for the analysis of differentially expressed proteins identified by mass spectrometry methods.Herein,we used a new strategy:data-dependent acquisition mode identification combined with label-free quantification by SWATH acquisition mode,to study the differentially expressed proteins in mouse liver cancer metastasis cells.A total of 1528 protein groups were identified,among which 1159 protein groups were quantified and 249 protein groups were observed as differentially expressed proteins(86 proteins up-regulated and 163 down-regulated).This method provides a commendable solution for the identification and quantification of differentially expressed proteins in biological samples.展开更多
Biomarke rs are required for the early detection,prognosis prediction,and monitoring of amyotrophic lateral sclerosis,a progressive disease.Proteomics is an unbiased and quantitative method that can be used to detect ...Biomarke rs are required for the early detection,prognosis prediction,and monitoring of amyotrophic lateral sclerosis,a progressive disease.Proteomics is an unbiased and quantitative method that can be used to detect neurochemical signatures to aid in the identification of candidate biomarke rs.In this study,we used a label-free quantitative proteomics approach to screen for substantially differentially regulated proteins in ten patients with sporadic amyotrophic lateral scle rosis compared with five healthy controls.Su bstantial upregulation of serum proteins related to multiple functional clusters was observed in patients with spo radic amyotrophic lateral sclerosis.Potential biomarke rs were selected based on functionality and expression specificity.To validate the proteomics profiles,blood samples from an additional cohort comprising 100 patients with sporadic amyotrophic lateral sclerosis and 100 healthy controls were subjected to enzyme-linked immunosorbent assay.Eight substantially upregulated serum proteins in patients with spora dic amyotrophic lateral sclerosis were selected,of which the cathelicidin-related antimicrobial peptide demonstrated the best discriminative ability between patients with sporadic amyotrophic lateral sclerosis and healthy controls(area under the curve[AUC]=0.713,P<0.0001).To further enhance diagnostic accuracy,a multi-protein combined discriminant algorithm was developed incorporating five proteins(hemoglobin beta,cathelicidin-related antimicrobial peptide,talin-1,zyxin,and translationally-controlled tumor protein).The algo rithm achieved an AUC of 0.811 and a P-value of<0.0001,resulting in 79%sensitivity and 71%specificity for the diagnosis of sporadic amyotrophic lateral scle rosis.Subsequently,the ability of candidate biomarkers to discriminate between early-stage amyotrophic lateral sclerosis patients and controls,as well as patients with different disease severities,was examined.A two-protein panel comprising talin-1 and translationally-controlled tumor protein effectively distinguished early-stage amyotrophic lateral sclerosis patients from controls(AUC=0.766,P<0.0001).Moreove r,the expression of three proteins(FK506 binding protein 1A,cathelicidin-related antimicrobial peptide,and hemoglobin beta-1)was found to increase with disease progression.The proteomic signatures developed in this study may help facilitate early diagnosis and monitor the progression of sporadic amyotrophic lateral sclerosis when used in co mbination with curre nt clinical-based parameters.展开更多
The presented work is to determine the mass fraction of angiotensinⅠ in solution by isotope dilution method,which is a pilot study organized by Consultative Committee for Amount of Substance.Firstly,the hydrolysis ti...The presented work is to determine the mass fraction of angiotensinⅠ in solution by isotope dilution method,which is a pilot study organized by Consultative Committee for Amount of Substance.Firstly,the hydrolysis time and hydrochloric acid addition volume ratio were optimized and the degradation of natural and isotope labeled proline,valine,leucine and phenylalanine during the hydrolysis process were investigated.Secondly,the mass fractions of proline,valine,isoleucine and phenylalanine in the angiotensinⅠhydrolysis solution was determined employing isotope dilution method with their reference materials as standards.Finally,the angiotensinⅠ mass fraction and associated uncertainty were calculated according to the amino acid mass fraction obtained,which was(75.2±2.6) μg/g.It showed that the result obtained in this paper has good equivalence with the average.It can be concluded that the isotope dilution method for peptide quantification is accurate and is suitable for peptide reference materials production.展开更多
目的通过所检测到的肽段丰度计算蛋白质丰度是蛋白质组学的一个重要部分。由于退化肽段的丰度可能由多个蛋白质提供,简单剔除退化肽段可消除这种不确定性并简化问题。但是由于退化肽段的信息没有被充分利用,蛋白质定量的准确性会受到影...目的通过所检测到的肽段丰度计算蛋白质丰度是蛋白质组学的一个重要部分。由于退化肽段的丰度可能由多个蛋白质提供,简单剔除退化肽段可消除这种不确定性并简化问题。但是由于退化肽段的信息没有被充分利用,蛋白质定量的准确性会受到影响,还可能显著降低可以定量的蛋白质规模。如何充分利用退化肽段的信息,提高蛋白定量的准确性和全面性,并且不会导致问题规模更为复杂,成为一个重要的问题。方法为了在不引入更多误差的情况下充分利用退化肽段来进行更准确的定量,本文提出一个基于误差最小化的方法(error-minimization-based quantification for protein,EMQ)。不同于以往的大多数算法,EMQ利用退化肽段中的信息,最大限度地将肽段层面信息还原到蛋白质层面,得到了更多的蛋白质定量结果并提高了结果的精度。结果多个实验数据上的表现证明本方法可以在较小的时间代价下获得更高的精度,并提高结果的规模。结论本文提出的基于误差最小化的方法可以快速准确地对大规模蛋白质组学问题进行定量。展开更多
High-throughput quantification with label-free methods has received considerable attention in electrospray ionization(ESI)-mass spectrometry(MS),but the manner by which MS signals respond to peptide concentration rema...High-throughput quantification with label-free methods has received considerable attention in electrospray ionization(ESI)-mass spectrometry(MS),but the manner by which MS signals respond to peptide concentration remains unclear in proteomics.We developed a new mathematical formula to describe the intrinsic log-log relationship between the MS intensity response and peptide concentration in an analytical ESI process.Experimental results showed that the calibration curve is fairly fit to the log-log formula with a linear dynamic range of approximate four to five orders of magnitude.However,we found that the ionization of analytical peptides can be severely suppressed by coexisting matrix peptides,such that the calibration curve can be poorly leveled off on both ends.Our study suggests that the interferences from coexisting matrix peptides should be reduced in the ESI process to use the log-log calibration curve successfully for the high-throughput quantification.展开更多
Food allergy is a growing health issue worldwide and the demand for sensitive,robust and high-throughput analytical methods is rising.In recent years,mass spectrometry-based methods have been established for multiple ...Food allergy is a growing health issue worldwide and the demand for sensitive,robust and high-throughput analytical methods is rising.In recent years,mass spectrometry-based methods have been established for multiple food allergen detection.In the present study,a novel method was developed for the simultaneous detection of almond,cashew,peanut,and walnut allergens in bakery foods using liquid chromatography–mass spectrometry.Proteins unique to these four ingredients were extracted,followed by trypsin digestion,quadrupole time-of-flight(Q-TOF)mass spectrometry and bioinformatics analysis.The raw data were processed by de-novo sequencing module plus PEAKS DB(database search)module of the PEAKS software to screen peptides specific to each nut species.The thermal stability and uniqueness of these candidate peptides were further verified using triple quadrupole mass spectrometry(QQQ-MS)in multiple reaction monitoring(MRM)mode.Each nut species was represented by four peptides,all of which were validated for label-free quantification(LFQ).Calibration curves were constructed with good linearity and correlation coefficient(r 2)greater than 0.99.The limits of detection(LODs)were determined to range from 0.11 to 0.31 mg/kg,and were compared with the reference doses proposed by Voluntary Incidental Trace Allergen Labelling(VITAL).The recoveries of the developed method in incurred bakery food matrices ranged from 72.5%to 92.1%with relative standard deviations(RSD)of<5.2%.The detection of undeclared allergens in commercial bakery food samples confirmed the presence of these allergens.In conclusion,this method provides insight into the qualitative and quantitative detection of trace levels of nut allergens in bakery foods.展开更多
基金supported by the Key Program of the National Natural Science Foundation of China(Grant No.:82130111)the National Natural Science Foundation of China(Grant No.:81803716)+1 种基金the Qi-Huang Chief Scientist Project of the National Administration of Traditional Chinese Medicine,China(2020)the SIMM-SHUTCM Traditional Chinese Medicine Innovation Joint Research Program,China(Grant No.:E2G809H).
文摘Pheretima,also called“earthworms”,is a well-known animal-derived traditional Chinese medicine that is extensively used in over 50 Chinese patent medicines(CPMs)in Chinese Pharmacopoeia(2020 edition).However,its zoological origin is unclear,both in the herbal market and CPMs.In this study,a strategy for integrating in-house annotated protein databases constructed from close evolutionary relationship-sourced RNA sequencing data from public archival resources and various sequencing algorithms(restricted search,open search,and de novo)was developed to characterize the phenotype of natural peptides of three major commercial species of Pheretima,including Pheretima aspergillum(PA),Pheretima vulgaris(PV),and Metaphire magna(MM).We identified 10,477 natural peptides in the PA,7,451 in PV,and 5,896 in MM samples.Five specific signature peptides were screened and then validated using synthetic peptides;these demonstrated robust specificity for the authentication of PA,PV,and MM.Finally,all marker peptides were successfully applied to identify the zoological origins of Brain Heart capsules and Xiaohuoluo pills,revealing the inconsistent Pheretima species used in these CPMs.In conclusion,our integrated strategy could be used for the in-depth characterization of natural peptides of other animal-derived traditional Chinese medicines,especially non-model species with poorly annotated protein databases.
基金support from the National Natural Science Foundation of China (Grant Nos. 20735004 & 20975101)the State Key Basic Research Program of China (Grant Nos. 2005CB522701 & 2007CB914102)+3 种基金the High Technology Research Pro-gram of China (Grant Nos. 2006AA02A309 & 2008ZX10002-017)the Knowledge Innovation Program of Chinese Academy of Sciences (Grant Nos. KJCX2.YW.HO9 & KSCX2-YW-R-079)the Knowledge Innova-tion Program of Dalian Institute of Chemical Physics to Zou HF and the China High Technology Research Program (Grant No. 2008ZX1002-020)the National Natural Science Foundation of China (Grant Nos. 20605022 & 90713017) to Ye ML
文摘Peptidomics draws more and more attention in discovering useful biomarkers for early diagnosis of disease. However, there is lack of efficient quantification strategy in peptidome analysis. In this study, a strategy with label-free quantification of the targeted endogenous peptides based on peak intensity using μUPLC-Q-TOF-MS/MS was developed for quantitative peptidome analysis of human serum. Different amounts of standard BSA tryptic digesting peptides were added into the same serum extracts for evaluation of the developed strategy, and it was observed that the average relative error of the targeted peptides was 6.42%, which was superior to the result obtained directly by commercially available software PLGS. It was also demonstrated that this quantification strategy could obviously increase the detection sensitivity of the peptide by DDA analysis. Then, this strategy was applied to comparatively analyze the peptides extracted from the serum of HCC or breast cancer patients and healthy individuals, respectively. Peptides with charge states up to 5 and molecular weight over 4000 can be reliably identified and quantified. This quantitative analysis method based on μUPLC-Q-TOF-MS/MS exhibited superior sensitivity than that by MALDI-TOF-MS commonly used in peptidome analysis. Finally, some interesting endogenous peptides related to corresponding diseases were successfully obtained.
文摘Pyroglutamyl(pGlu)peptides are formed from intramolecular cyclization of glutamine or glutamic acid residue at the N-terminal position of peptides.This process can occur endogenously or during processing of foods containing the peptides.Some factors such as heat,high pressure and enzymatic modifications contribute to pGlu formation.pGlu peptides are thought to have different characteristics,especially bitter and umani tastes,and thus can affect the sensory properties of foods that contain them.Moreover,some health-promoting properties have been reported for pGlu peptides,including hepatoprotective,antidepressant and anti-inflammatory activities.However,the role of pGlu residue in the peptide bioactivity is not completely established,although the hydrophobic-lactam ring is thought to enhance the peptide stability against degradation by gastrointestinal proteases.This review discusses the occurrence and formation of pGlu peptides in foods,their quantification,sensory and biological properties,and prospects in food applications.
基金This work was supported by grants from the National Natural Science Foundation of China(Grant No.31800601).
文摘Studying the genetic basis and regulatory mechanism of chrysanthemum lateral bud outgrowth is of great significance for reduction the production cost of cut chrysanthemum.To clarify the molecular basis of lateral bud elongation after removal of apical dominance in chrysanthemum,label-free quantification analysis was used to analyze the proteome changes after apical bud removal.Quantitative real-time PCR(qPCR)was used to analyze the changes in the expression of three plant hormone-related genes.A total of 440 differentially expressed proteins were successfully identified at three time points during the lateral bud elongation.The number of differentially expressed proteins in the three stages(24 h/0 h,48 h/0 h,48 h/24 h)were 219,332,and 97,respectively.The difference in expressed proteins in the three comparison stages mainly involves RNA processing and modification;translation,ribosomal structure and biogenesis;Posttranslational modification,protein turnover,and chaperones.Path analysis showed that there was various physiological activities in the process of lateral bud dormancy breaking and elongation,which involved energy metabolism,biosynthesis,signal transduction and stress response in the growth process of lateral buds.qPCR indicated that the expression of cytokinin synthesis related gene was significantly increased after the removal of apical dominance,while the expression of strigolactones synthesis related gene experiences a dramatic fall to promote the development of the lateral buds.However,there was a drop before a slight increase in the expression of the auxin synthesis related gene,which was mainly due to the removal of apical dominance that led to the loss of indoleacetic acid in the main stem.However,with formation of the new apical source,indoleacetic acid can be released again.
基金financial support from the National Basic Research Program of China(2012CB910602,92013CB911200)the National Natural Science Foundation of China(2100507,21235005)+1 种基金the Creative Research Group Project by NSFC(21021004)the National High Technology Research and Development Program of China(2012AA020202)
文摘Label-free quantification is a valuable tool for the analysis of differentially expressed proteins identified by mass spectrometry methods.Herein,we used a new strategy:data-dependent acquisition mode identification combined with label-free quantification by SWATH acquisition mode,to study the differentially expressed proteins in mouse liver cancer metastasis cells.A total of 1528 protein groups were identified,among which 1159 protein groups were quantified and 249 protein groups were observed as differentially expressed proteins(86 proteins up-regulated and 163 down-regulated).This method provides a commendable solution for the identification and quantification of differentially expressed proteins in biological samples.
基金supported by the grants from Shanghai Shuguang Plan Project,No.18SG15(to SC)Shanghai Outstanding Young Scholars Project+2 种基金Shanghai Talent Development Project,No.2019044(to SC)Medical-engineering cross fund of Shanghai Jiao Tong University,No.YG2022QN009(to QZ)the National Natural Science Foundation of China,No.82201558(to QZ)。
文摘Biomarke rs are required for the early detection,prognosis prediction,and monitoring of amyotrophic lateral sclerosis,a progressive disease.Proteomics is an unbiased and quantitative method that can be used to detect neurochemical signatures to aid in the identification of candidate biomarke rs.In this study,we used a label-free quantitative proteomics approach to screen for substantially differentially regulated proteins in ten patients with sporadic amyotrophic lateral scle rosis compared with five healthy controls.Su bstantial upregulation of serum proteins related to multiple functional clusters was observed in patients with spo radic amyotrophic lateral sclerosis.Potential biomarke rs were selected based on functionality and expression specificity.To validate the proteomics profiles,blood samples from an additional cohort comprising 100 patients with sporadic amyotrophic lateral sclerosis and 100 healthy controls were subjected to enzyme-linked immunosorbent assay.Eight substantially upregulated serum proteins in patients with spora dic amyotrophic lateral sclerosis were selected,of which the cathelicidin-related antimicrobial peptide demonstrated the best discriminative ability between patients with sporadic amyotrophic lateral sclerosis and healthy controls(area under the curve[AUC]=0.713,P<0.0001).To further enhance diagnostic accuracy,a multi-protein combined discriminant algorithm was developed incorporating five proteins(hemoglobin beta,cathelicidin-related antimicrobial peptide,talin-1,zyxin,and translationally-controlled tumor protein).The algo rithm achieved an AUC of 0.811 and a P-value of<0.0001,resulting in 79%sensitivity and 71%specificity for the diagnosis of sporadic amyotrophic lateral scle rosis.Subsequently,the ability of candidate biomarkers to discriminate between early-stage amyotrophic lateral sclerosis patients and controls,as well as patients with different disease severities,was examined.A two-protein panel comprising talin-1 and translationally-controlled tumor protein effectively distinguished early-stage amyotrophic lateral sclerosis patients from controls(AUC=0.766,P<0.0001).Moreove r,the expression of three proteins(FK506 binding protein 1A,cathelicidin-related antimicrobial peptide,and hemoglobin beta-1)was found to increase with disease progression.The proteomic signatures developed in this study may help facilitate early diagnosis and monitor the progression of sporadic amyotrophic lateral sclerosis when used in co mbination with curre nt clinical-based parameters.
文摘The presented work is to determine the mass fraction of angiotensinⅠ in solution by isotope dilution method,which is a pilot study organized by Consultative Committee for Amount of Substance.Firstly,the hydrolysis time and hydrochloric acid addition volume ratio were optimized and the degradation of natural and isotope labeled proline,valine,leucine and phenylalanine during the hydrolysis process were investigated.Secondly,the mass fractions of proline,valine,isoleucine and phenylalanine in the angiotensinⅠhydrolysis solution was determined employing isotope dilution method with their reference materials as standards.Finally,the angiotensinⅠ mass fraction and associated uncertainty were calculated according to the amino acid mass fraction obtained,which was(75.2±2.6) μg/g.It showed that the result obtained in this paper has good equivalence with the average.It can be concluded that the isotope dilution method for peptide quantification is accurate and is suitable for peptide reference materials production.
文摘目的通过所检测到的肽段丰度计算蛋白质丰度是蛋白质组学的一个重要部分。由于退化肽段的丰度可能由多个蛋白质提供,简单剔除退化肽段可消除这种不确定性并简化问题。但是由于退化肽段的信息没有被充分利用,蛋白质定量的准确性会受到影响,还可能显著降低可以定量的蛋白质规模。如何充分利用退化肽段的信息,提高蛋白定量的准确性和全面性,并且不会导致问题规模更为复杂,成为一个重要的问题。方法为了在不引入更多误差的情况下充分利用退化肽段来进行更准确的定量,本文提出一个基于误差最小化的方法(error-minimization-based quantification for protein,EMQ)。不同于以往的大多数算法,EMQ利用退化肽段中的信息,最大限度地将肽段层面信息还原到蛋白质层面,得到了更多的蛋白质定量结果并提高了结果的精度。结果多个实验数据上的表现证明本方法可以在较小的时间代价下获得更高的精度,并提高结果的规模。结论本文提出的基于误差最小化的方法可以快速准确地对大规模蛋白质组学问题进行定量。
基金supported by the National Basic Research Program of China(2010CB912700,2013CB911201,2011CB910600)the National High Technology Research and Development Program of China(2012AA020203,2014AAQ00361)the National Natural Science Foundation of China(21227805)
文摘High-throughput quantification with label-free methods has received considerable attention in electrospray ionization(ESI)-mass spectrometry(MS),but the manner by which MS signals respond to peptide concentration remains unclear in proteomics.We developed a new mathematical formula to describe the intrinsic log-log relationship between the MS intensity response and peptide concentration in an analytical ESI process.Experimental results showed that the calibration curve is fairly fit to the log-log formula with a linear dynamic range of approximate four to five orders of magnitude.However,we found that the ionization of analytical peptides can be severely suppressed by coexisting matrix peptides,such that the calibration curve can be poorly leveled off on both ends.Our study suggests that the interferences from coexisting matrix peptides should be reduced in the ESI process to use the log-log calibration curve successfully for the high-throughput quantification.
基金the Key Science and Technology Program of the Market Supervision Administration of Jiangsu Province(No.KJ196035)the Key Science and Technology Program of the Market Supervision Administration of Nanjing(No.KJ2019043),China.
文摘Food allergy is a growing health issue worldwide and the demand for sensitive,robust and high-throughput analytical methods is rising.In recent years,mass spectrometry-based methods have been established for multiple food allergen detection.In the present study,a novel method was developed for the simultaneous detection of almond,cashew,peanut,and walnut allergens in bakery foods using liquid chromatography–mass spectrometry.Proteins unique to these four ingredients were extracted,followed by trypsin digestion,quadrupole time-of-flight(Q-TOF)mass spectrometry and bioinformatics analysis.The raw data were processed by de-novo sequencing module plus PEAKS DB(database search)module of the PEAKS software to screen peptides specific to each nut species.The thermal stability and uniqueness of these candidate peptides were further verified using triple quadrupole mass spectrometry(QQQ-MS)in multiple reaction monitoring(MRM)mode.Each nut species was represented by four peptides,all of which were validated for label-free quantification(LFQ).Calibration curves were constructed with good linearity and correlation coefficient(r 2)greater than 0.99.The limits of detection(LODs)were determined to range from 0.11 to 0.31 mg/kg,and were compared with the reference doses proposed by Voluntary Incidental Trace Allergen Labelling(VITAL).The recoveries of the developed method in incurred bakery food matrices ranged from 72.5%to 92.1%with relative standard deviations(RSD)of<5.2%.The detection of undeclared allergens in commercial bakery food samples confirmed the presence of these allergens.In conclusion,this method provides insight into the qualitative and quantitative detection of trace levels of nut allergens in bakery foods.
