基于生物质谱的蛋白质组学分析大西洋鲑中生物标志性肽段及过敏原特异性肽段  

Analysis of Biomarker Peptides and Allergen Specific Peptide in Salmo Salar by Proteomics Based on Biomass Spectrometry

作　　者：罗娇依 郑越男 刘彤彤 曹进[1] 郭亚辉[3] 孙姗姗[1] LUO Jiaoyi;ZHENG Yuenan;LIU Tongtong;CAO Jin;GUO Yahui;SUN Shanshan(Key Laboratory of Food Quality and Safety for State Market Regulation,National Institutes for Food and Drug Control,Beijing 100050,China;Songzi Center for Inspection and Test,Songzi 434200,China;State Key Laboratory of Food Science and Technology,School of Food Science and Technology,Jiangnan University,Wuxi 214122,China)

机构地区：[1]中国食品药品检定研究院国家市场监管重点实验室(食品质量与安全),北京100050 [2]松滋市公共检验检测中心,松滋434200 [3]江南大学食品学院食品科学与技术国家重点实验室,无锡214122

出　　处：《理化检验（化学分册）》2023年第1期1-12,共12页Physical Testing and Chemical Analysis(Part B:Chemical Analysis)

基　　金：中国食品药品检定研究院中青年发展研究基金(2020C4)。

摘　　要：基于蛋白质组学自下而上研究策略,采用纳升液相色谱-四极杆-静电场轨道阱高分辨质谱联用法(Nano-UHPLC-Q/Exactive-HRMS)和超高效液相色谱-串联质谱法(UHPLC-MS/MS)筛选、鉴定大西洋鲑中生物标志性肽段和过敏原β-小清蛋白特异性肽段,并提出了UHPLC-MS/MS测定其含量的方法。称取5 g处理好的样品,加入10 mL正己烷除脂,离心,于沉淀中加入5 mL磷酸盐缓冲溶液(提取剂),涡旋,于60℃水浴加热30 min,再用20 mL冷丙酮富集、纯化,得到的蛋白提取液经胰蛋白酶酶解后,采用Nano-UHPLC-Q/Exactive-HRMS初步筛选大西洋鲑生物标志性肽段与过敏原β-小清蛋白特异性肽段,用UHPLC-MS/MS对初筛的肽段进行多反应监测(MRM)模式验证。在酶解后的溶液中加入内标,采用UHPLC-MS/MS测定,内标法定量。结果显示:筛选出3条可用于定量的大西洋鲑生物标志性肽段VAVNVEETK、VAPLSEDFK、LTEIQSLER和1条过敏原β-小清蛋白特异性肽段TFFHTIGFASK;VAVNVEETK、VAPLSEDFK、LTEIQSLER标准曲线的线性范围在5.00μmol·L^(-1)以内,TFFHTIGFASK标准曲线的线性范围在10.00μmol·L^(-1)以内,检出限(3S/N)为0.32~1.55μg·g^(-1);空白样品中4条肽段的加标回收率为90.2%~105%,测定值的相对标准偏差(n=6)为1.5%~4.6%;方法用于测定大西洋鲑贮存过程中VAVNVEETK、VAPLSEDFK、LTEIQSLER、TFFHTIGFASK的含量,LTEIQSLER和TFFHTIGFASK含量没有发生明显变化,而VAVNVEETK和VAPLSEDFK含量随着贮存天数的延长逐渐降低,其中VAPLSEDFK适用于大西洋鲑新鲜度评价。Based on the bottom-up research strategy of proteomics, nano liquid chromatography-quadrupole-electrostatic field orbital trap high resolution mass spectrometry(Nano-UHPLC-Q/Exactive-HRMS) and ultra-high performance liquid chromatography-tandem mass spectrometry(UHPLC-MS/MS) were used to screen and identify biomarker peptides and allergen β-parvalbumin specific peptide in Salmo salar, and a method for the determination of them by UHPLC-MS/MS was proposed. The processed sample(5 g) was taken, and 10 mL of n-hexane was added for grease removal. After centrifugation, 5 mL of phosphate buffer solution(as extractant) was added into the precipitation. The mixture was whirled and heated in water bath at 60 ℃ for 30 min, and 20 mL of cold acetone was used for enrichment and purification. The obtained protein extract was hydrolyzed by trypsin, and Nano-UHPLC-Q/Exactive HRMS was used to preliminary screen the biomarker peptides and the specific peptide of allergen β-parvalbumin in Salmo salar, and then UHPLC-MS/MS was used to verify the screened peptides with multiple reaction monitoring(MRM) mode. Internal standards were added into the solution after enzymolysis, and UHPLC-MS/MS was used for determination, with internal standard method for quantitative analysis. It was shown that 3 biomarker peptides VAVNVEETK, VAPLSEDFK, LTEIQSLER and one allergen β-parvalbumin specific peptide TFFHTIGFASK were screened for quantification. The linear ranges of standard curves for VAVNVEETK, VAPLSEDFK and LTEIQSLER were within 5.00 μmol·L^(-1), and that for TFFHTIGFASK was within 10.00 μmol·L^(-1), with detection limits(3S/N) in the range of 0.32^(-1).55 μg·g^(-1). The recoveries of 4 peptides in the blank samples ranged from 90.2% to 105%, and RSDs(n=6) of the determined values were in the range of 1.5%-4.6%. This method was applied to determination of VAVNVEETK, VAPLSEDFK, LTEIQSLER and TFFHTIGFASK during the storage of Salmo salar. The contents of LTEIQSLER and TFFHTIGFASK did not change significantly, the contents of VAVNVE

关 键 词：大西洋鲑 生物标志性肽段 β-小清蛋白 特异性肽段 蛋白质组学 

分 类 号：O657.63[理学—分析化学]