以合成肽段ALTDAETK为定量特征肽段,SDFIEEDELK及LFLQNFAFSASAR为定性肽段,建立牙鲆中小清蛋白高效液相色谱-串联质谱的检测方法。同时对蛋白提取液、碘代乙酰胺浓度、酶/蛋白质比例、酶解时间在内的前处理条件和肽段质谱参数条件进行了优化。结果显示,ALTDAETK肽段在0.005~10 mg/L线性关系良好(R2>0.999),小清蛋白定量限 2.74 mg/kg;以大菱鲆为空白基质,重组小清蛋白的平均回收率为95%～102%,相对标准偏差(RSD )小于7%,重复性好。该文首次实现了用HPLC-MS/MS方法精确定量检测牙鲆中主要过敏原PV。
A method using high performance chromatography tandem mass spectrometry/mass spectrometry(HPLC-MS/MS)detecting parvalbumin in Paralichthys olivaceus was established. ALTDAETK was used as a quantitative marker peptide, SDFIEEDELK and LFLQNFAFSASAR were used as qualitative marker peptides. The pretreatment conditions including protein extracts, indole-3-acetic acid (IAA) concentration, enzyme/protein ratio, and enzymatic hydrolysis time, as well as the instrumental conditions for mass spectrometry parameters used for three marker peptides were optimized. The results showed that ALTDAETK had a good linear relationship in a range of 0.005-10 mg/L (R2>0.999). The quantification limit of parvalbumin was 2.74 mg/kg. Took Psetta maxima as a blank, the average recovery rate of recombinant parvalbumin was 94.8%-102.0%, and the relative standard deviation (RSD) was below 7% with a good repeatability. It was the first time that parvalbumin was detected in Paralichthys olivaceus accurately and quantitatively using HPLC-MS/MS.
[1] 农小献,宾石玉,蒙涛.等.小清蛋白研究进展[J].生物技术通讯,2011,22(6):887-891.
[2] MONACI L,VISCONTI A.Mass spectrometry-based proteomics methods for analysis of food allergens[J].TrAC Trends in Analytical Chemistry,2009,28(5):581-591.
[3] 詹丽娜,陈沁,古淑青,等.超高效液相色谱－四极杆／静电场轨道阱高分辨质谱检测食品中的牛奶过敏原酪蛋白[J].色谱,2017,35(4):405-412.
[4] JOHNSON P E,BAUMGARTNER S,ALDICK T,et al.Current perspectives and recommendations for the development of mass spectrometry methods for the determination of allergens in foods[J].Journal of Aoac.International,2011,94(4):1 026-1 033.
[5] WANG Yang,WU Liqing,DUAN Fei,et al. Development of an SI-Traceable HPLC-isotope dilution mass spectrometry method to quantify β-Lactoglobulin in milk powders[J]. Journal of Agricultural & Food Chemistry,2014,62(14):3 073-3 080.
[6] CARRERA M,CAÑAS B,GALLARDO J M.Rapid direct detection of the major fish allergen,parvalbumin,by selected MS/MS ion monitoring mass spectrometry[J].Journal of Proteomics,2012,75(11):3 211-3 220.
[7] 孙一帆,黄建芳,向军俭.等.凡纳滨对虾过敏原精氨酸激酶的质谱鉴定及其免疫交叉反应研究[J].食品科学,2015,36(1):170-173.
[8] KORTE R,BROCKMEYER J.MRM3-based LC-MS multi-method for the detection and quantification of nut allergens[J].Analytical and Bioanalytical Chemistry,2016,408(27):1 845-1 855.
[9] HUSCHEK G,BÔNICK J,LÔWENSTEIN Y,et al.Quantification of allergenic plant traces in baked products by targeted proteomics using isotope marked peptides[J].LWT - Food Science and Technology,2016,74:286-293.
[10] SETTLAGE S B,EBLE J E,BHANUSHALI J K,et al.Validation parameters for quantitating specific proteins using ELISA or LC-MS/MS: Survey results[J].Food Analytical Methods,2017,10(5):1 339-1 348.
[11] 张舒亚,于翠,宋青,等.食品过敏原大豆P34蛋白基因的实时荧光PCR检测[J].中国油料作物学报,2012,34(6):661-665.
[12] CAI Qiufeng,WANG Xichang,LIU Guangming,et al.Development of a monoclonal antibody-based competitive enzyme;linked-immunosorbent assay (c-ELISA) for quantification of silver carp parvalbumin[J].Food Control,2013,29(1):241-247.
[13] 蔡秋凤,王锡昌,郭城,等. 加工处理对鲢小清蛋白免疫活性的影响[J].水产学报,2013,37(2):297-302.
[14] COX J,MANN M.MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification[J].Nature Biotechnology,2008,26(12):1 367-1 372.
[15] HOFFMANN B,MUNCH S,SCHWAGELE F,et al.A sensitive HPLC-MS/MS screening method for the simultaneous detection of lupine,pea,and soy proteins in meat products[J].Food Control,2017,71:200-209.
[16] WU Lisha,LIN Haixin,WANG Guoying,et al.Comparative study on the allergenicity of different Litopenaeus vannamei extract solutions[J].Journal of Ocean University of China,2014,13(1):157-162.
[17] YANG Z,ATTYGALLE A B.LC/MS characterization of undesired products formed during iodoacetamide derivatization of sulfhydryl groups of peptides[J].Journal of Mass Spectrometry Jms,2010,42(2):233-243.
[18] NORRGRAM J,WILLIAMS T L,WOOLFITT A R,et al. Optimization of digestion parameters for protein quantification[J].Analytical Biochemistry,2009,393(1):48-55.
