《质谱分析技术》PPT课件

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1、用于蛋白质鉴定的两种基本的质谱分析技术1）MALDI-TOFMSMatrix-AssistedLaserDesorptionIonization/基质辅助激光解吸附离子化Time-Of-Flightmassspectrometry飞行时间PMF:peptidemassfingerprinting肽质量指纹谱MW:800-300,0002）SELDI-TOFMSSurfaceEnhancedLaserDesorption/Ionization表面增强激光解吸附离子化MW:2,000-50,0003）MS/MSTandemMS串连质谱技术MW:200-3,0001）MALDI-TOF

2、MS（原理示意图）SourceLength=sField-freedriftzoneLength=DEd=0MicrochannelplatedetectorExtractiongrid(sourcevoltage-Vs)UV(337nm)Detectorgrid-VsPulsevoltageAnalyte/matrix用MALDI-TOFMS和数据库鉴定细菌蛋白GroELPMF肽质量指纹谱鉴定蛋白质例子MyoglobinGLSDGEWQQVLNVWGKVEADIAGHGQEVLIRLFTGHPETLEKFDKFKHLKTEAEMKASEDLKKHGTVVLTALGGILKKKG

3、HHEAELKPLAQSHATKHKIPIKYLEFISDAIIHVLHSKHPGDFGADAQGAMTKALELFRNDIAAKYKELGFQGMyoglobin用胰蛋白酶限制酶切后产生的肽段（从碱性氨基酸的羧基端切断）MyoglobinGLSDGEWQQVLNVWGKVEADIAGHGQEVLIRLFTGHPETLEKFDKFKHLKTEAEMKASEDLKKHGTVVLTALGGILKKKGHHEAELKPLAQSHATKHKIPIKYLEFISDAIIHVLHSKHPGDFGADAQGAMTKALELFRNDIAAKYKELGFQG胰蛋白酶酶切Myoglobin产生的肽段

4、及其分子量1811.90GLSDGEWQQVLNVWGK1606.85VEADIAGHGQEVLIR1271.66LFTGHPETLEK1378.83HGTVVLTALGGILK1982.05KGHHEAELKPLAQSHATK1853.95GHHEAELKPLAQSHATK1884.01YLEFISDAIIHVLHSK1502.66HPGDFGADAQGAMTK748.43ALELFR实际质谱分析所得的肽质量指纹谱GLSDGEWQQVLNVWGKVEADIAGHGQEVLIRLFTGHPETLEKHGTVVLTALGGILKKGHHEAELKPLAQSHATKGHHEAELKP

5、LAQSHATKYLEFISDAIIHVLHSKHPGDFGADAQGAMTKALELFR2）SELDI-TOFMS（原理示意图）EAM:能量吸收分子SELDI-TOFMS分析使用的各种蛋白质芯片SELDI-TOFMA检测结果与处理实例：鼻咽癌病人血清标志物检测卵巢癌细胞培养液上清中分泌蛋白的SELDI-TOFMS检测分析2）MS/MS串连质谱分析原理示意图限制酶切蛋白质为肽段EnzymaticDigestandFractionation第一步MS分离各个肽段第二步MS对其中的一个肽段进行氨基酸序列分析MS/MS碰撞诱导解离CID中多肽分子结构中键断裂的多种可能性-HN-CH-

6、CO-NH-CH-CO-NH-RiCH-R’biyn-iyn-i-1bi+1R”i+1i+1aixn-icizn-i控制CID过程中的能量，使多肽主要从最弱的键---肽键处断裂产生N-端b系列和C-端y系列肽段-HN-CH-CO-NH-CH-CO-NH-RiRi+1biyn-iyn-i-1bi+1串联质谱氨基酸序列分析例子肽段SGFLEEDELK在理想条件下可生成的b、y系列离子MWionionMW88b1SGFLEEDELKy91080145b2SGFLEEDELKy81022292b3SGFLEEDELKy7875405b4SGFLEEDELKy6762534b5SGFLEE

7、DELKy5633663b6SGFLEEDELKy4504778b7SGFLEEDELKy3389907b8SGFLEEDELKy22601020b9SGFLEEDELKy1147S=serine87.08G=glycine57.05F=phenylalanine147.18L=leucine113.16E=glutamate129.12D=aspartate115.09K=lysine128.17先找出y系列离子K1166L1020E907D778E663E534L405F29