基于特征肽段的液相色谱-质谱技术鉴定胶原蛋白的物种来源

Identification of Species Origin of Collagen Based on Liquid Chromatography-Mass Spectrometry with Peptide Markers

建立了一种基于特征肽段的液相色谱-质谱技术鉴定胶原蛋白物种来源的方法。样品经蛋白提取,还原,烷基化,胰蛋白酶消化后,采用Eksigent C18色谱柱(75μm×150 mm,3μm)分离,用流动相0. 1%甲酸水-乙腈溶液(98∶2)和0. 1%甲酸乙腈-水溶液(98∶2)梯度洗脱,在正离子模式下,通过纳升电喷雾四极杆飞行时间质谱进行检测,数据经Protein PilotTM软件及blast分析,筛选出潜在的特征肽段。消化后的样品再采用Eclipse Plus C18色谱柱(2. 1 mm×100 mm,1. 8μm)分离,用流动相乙腈和1%甲酸水溶液梯度洗脱,在正离子模式下,通过电喷雾四极杆/线性离子阱串联质谱的多反应监测触发增强子离子扫描模式进行检测,进一步确认肽段的特异性。最终筛选并确证了3种猪源性胶原蛋白特征肽段,4种牛源性胶原蛋白特征肽段,1种羊源性胶原蛋白特征肽段。所筛选的特征肽段具有良好的耐热性,可为动物源性胶原蛋白鉴定提供一种特异性强、准确可靠的检测方法。

A liquid chromatography-mass spectrometric method was established for the identification of the species origin of collagen based on peptide markers.The sample was determined by nano electrospray ionization quadrupole time-of-flight mass spectrometry in positive mode to obtain peptide mass spectrometry information after protein extraction,reduction,alkylation,tryptic digestion and followed by the separation on an Eksigent C 18 column(75μm×150 mm,3μm)by gradient elution with water-acetonitrile(98∶2)containing 0.1%formic acid and acetonitrile-water(98∶2)containing 0.1%formic acid as mobile phases.Potential peptide markers were screened by data analysis with ProteinPilot TM and blast.Then the digested sample was separated on an Eclipse Plus C 18 column(2.1 mm×100 mm,1.8μm)by gradient elution with acetonitrile-water containing 0.1%formic acid as mobile phase.The specificity of peptide markers was further confirmed by electrospray ionization quadrupole/linear ion trap tandem mass spectrometry in positive mode under multiple reaction monitoring information dependent acquisition enhanced product ion(MRM-IDA-EPI)scanning mode.Finally,three pig collagen peptide markers,four bovine collagen peptide markers and one sheep collagen peptide marker were screened and identified.These peptide markers have a good heat resistance,and could provide a specific,accurate and reliable method for the identification on species origin of collagen.