STRUCTURAL CHARACTERIZATION OF PROTEIN TRYPTIC PEPTIDES VIA LIQUID-CHROMATOGRAPHY MASS-SPECTROMETRY AND COLLISION-INDUCED DISSOCIATION OF THEIR DOUBLY CHARGED MOLECULAR-IONS
STRUCTURAL CHARACTERIZATION OF PROTEIN TRYPTIC PEPTIDES VIA LIQUID-CHROMATOGRAPHY MASS-SPECTROMETRY AND COLLISION-INDUCED DISSOCIATION OF THEIR DOUBLY CHARGED MOLECULAR-IONS
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DOI：
10.1021/ac00013a003
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发表时间：
1991-07-01
影响因子：
7.4
通讯作者：
HENION, JD
中科院分区：
文献类型：
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作者：
COVEY, TR;HUANG, EC;HENION, JD
在电喷雾电离过程中，通过场辅助离子蒸发机制形成多个带电分子离子，使大气压电离四极杆质谱仪系统能够用于表征生物学上重要的肽。介绍了通过气动辅助电喷雾（离子喷雾）界面直接实施高效液相色谱（HPLC）来确定色氨酸肽分子量的新策略。示例使用微孔（1.0 mm）和标准孔（4.6 mm）内径柱的LC/MS分子量测定蛋氨酸-人生长激素（met-hGH）中色氨酸肽。将50至75 pmol的色氨酸消化液注射到1 mm i.d的HPLC柱上，提供全扫描LC/MS或LC/MS/MS结果，而无需柱后分离流出物。当使用标准4.6 mm的HPLC柱时，采用20:1的柱后分离，需要注射1至5 nmol的色氨酸消化液以获得全扫描LC/MS或LC/MS/MS结果。碰撞诱导解离（CID）质谱由“输注”或在线LC/MS/MS分析产生，在电喷雾条件下，丰富的双带电离子在胰蛋白酶肽中占主导地位，为met-hGH和人血红蛋白胰蛋白酶消化提供了结构上有用的序列信息。在样品输注过程中使用的质谱仪扫描速率较慢，比在线LC/MS或LC/MS/MS提供更准确的质量分配，但后者的在线实验排除了由基质或组分干扰引起的模糊性。然而，在某些情况下，非常弱的CID产物离子妨碍了仅基于CID数据的完整的色氨酸结构表征。在线LC/MS/MS分析了人血红蛋白正常β链的胰蛋白酶消化产物，提供了足够的重叠结构信息来推断具有代表性的胰蛋白酶片段的序列。这种方法提供了表征这些重要生物化合物的有效手段。
The formation of multiply charged molecular ions via the field-assisted ion evaporation mechanism during electrospray ionization enables the use of an atmospheric pressure ionization quadrupole mass spectrometer system for characterizing biologically important peptides. The straightforward implementation of high-performance liquid chromatography (HPLC) into this new strategy to determine the molecular weight of tryptic peptides via the pneumatically assisted electrospray (ion spray) interface is presented. Examples utilizing both microbore (1.0 mm) and standard bore (4.6 mm) inside diameter columns are shown for the LC/MS molecular weight determination of tryptic peptides in methionyl-human growth hormone (met-hGH). Injected levels from 50 to 75 pmol of tryptic digest onto 1 mm i.d. HPLC columns provided full-scan LC/MS or LC/MS/MS results without postcolumn splitting of the effluent. When standard 4.6 mm i.d. HPLC columns were used, a 20:1 postcolumn split was utilized, which required from 1 to 5 nmol of injected tryptic digest for full-scan LC/MS or LC/MS/MS results. Collision-induced dissociation (CID) mass spectra resulting from either "infusion" or on-line LC/MS/MS analysis of the abundant doubly charged ions that predominate for tryptic peptides under electrospray conditions provided structurally useful sequence information for met-hGH and human hemoglobin tryptic digests. The slower mass spectrometer scan rate used during infusion of sample provides more accurate mass assignments than on-line LC/MS or LC/MS/MS, but the latter on-line experiments preclude ambiguities caused by matrix or component interferences. However, in some instances very weak CID product ions preclude complete tryptic peptide structural characterization based upon the CID data alone. The on-line LC/MS/MS analysis of the tryptic digest from human hemoglobin normal beta-chain provided sufficient over-lapping structural information to deduce the sequence of a representative tryptic fragment. This approach provides an effective means of characterizing these biologically important compounds.