反相液相色谱-串联质谱法鉴定油菜蜂花粉中的蛋白质及活性肽  

Application of reversed-phase liquid chromatography-tandem mass spectrometryin the identification of protein and bioactivity peptides from rape bee pollen

作　　者：郭静[1,2] 晏嘉泽[2] 郭明[1] 靳艳[2] 

机构地区：[1]大连大学环境与化学工程学院,辽宁大连116622 [2]中国科学院大连化学物理研究所,中国科学院分离分析化学重点实验室,辽宁大连116023

出　　处：《色谱》2014年第3期284-289,共6页Chinese Journal of Chromatography

基　　金：东北特色生物资源综合利用项目(2013SYHZ0020)

摘　　要：基于鸟枪法蛋白质组学分析方法,使用反相液相色谱-串联质谱(RPLC-MS/MS)系统分析油菜蜂花粉蛋白质的胰蛋白酶酶解产物,结合数据库检索,共鉴定到353条肽段。鉴定到的肽段所归属的蛋白质中有239个蛋白质可检索到其分子生物学功能,主要功能为结合活性、酶活性、运输活性、抑制活性等。根据血管紧张素转化酶(ACE)抑制肽活性与多肽构效之间的关系,从鉴定到的肽段中筛选并适当修饰后得到5条可能具有ACE抑制活性的肽段,化学合成肽段后进行了活性验证。结果表明5条肽段均具有良好的活性,其中肽段AELDIVLALF和LAVNLIPFP表现出较高的ACE抑制活性,半数抑制浓度(IC50)分别为(10.65±0.50)μmol/L和(23.66±1.08)μmol/L。该方法速度快,成本低,大大缩短了鉴定周期,达到了高通量筛选生物活性肽的目的。Based on the shotgun proteomic method,rape bee pollen protein was prepared with ultrasonic extraction and digested by trypsin,then separated and sequenced by reversed-phase liquid chromatography-tandem mass spectrometry（ RPLC-MS / MS）,followed by protein data-base searching. After the above analysis,353 peptides were identified and the molecular biolog-ical functions of 239 proteins have been known. The identified molecular biological functions of these proteins mainly included binding activity,enzyme activity,transporter activity,inhibitor activity and so on. Five peptides were obtained after the screening and appropriate amino acid modification among the identified 353 peptides,according to the relationship between the sequence structure of the bioactivity peptide and angiotensin converting enzyme（ ACE）inhibi-tor activity. The five peptides were speculated to have ACE inhibitor activity and synthesized to detect ACE inhibitor activity. The results showed that all of the five peptides had good ACE inhibitor activity. The peptides of AELDIVLALF and LAVNLIPFP among the five peptides dis-played especially good ACE inhibition with half maximal inhibitory concentration（ IC 50 ） of （10. 65＆#177;0. 50）μmol / L and（23. 66＆#177;1. 08）μmol / L,respectively. This method is rapid,low-cost and achieves the goal of high-throughput screening of bioactivity peptides that greatly shorten the period of identification compared with traditional methods.

关 键 词：反相液相色谱-串联质谱 蛋白质 血管紧张素转化酶抑制肽 油菜蜂花粉 REVERSED-PHASE liquid chromatography-tandem mass SPECTROMETRY ( RPLC-MS MS) 

分 类 号：O658[理学—分析化学]