Protein digest analysis by pressurized capillary electrochromatography using an ion trap storage/reflectron time of flight mass detector
Protein digest analysis by pressurized capillary electrochromatography using an ion trap storage/reflectron time of flight mass detector
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DOI：
10.1021/ac970183g
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发表时间：
1997-08-01
影响因子：
7.4
通讯作者：
Lubman, DM
中科院分区：
文献类型：
--
作者：
Wu, JT;Huang, PQ;Lubman, DM
将加压毛细管电色谱（pCEC）与离子阱储存/反射飞行时间质谱联用，用于肽类混合物和蛋白质类化合物的分析。由于pCEC具有较好的电渗流特性和较小的填充材料，在这些实验中使用补充压力，其抑制气泡形成并且还允许使用电场调节肽的洗脱。在这项工作中，已经成功地进行了六肽混合物的快速分离。在6cm长的色谱柱上，通过适当调节外加电压和辅助压力，牛细胞色素c的胰蛋白酶消化物在14 min左右就能完全分离。此外，用该系统分析了鸡卵清蛋白的胰蛋白酶消化物等相对复杂的蛋白质酶，在17 min内就能分辨出总离子流色谱中的20多个峰。使用离子阱存储/反射飞行时间质谱仪作为在线检测器，通过明确识别共洗脱组分进一步提高了pCEC的分辨能力。在这些实验中，成功地将飞行时间质量分析仪的非扫描特性和离子阱的离子信号集成能力结合起来，以提供快速和灵敏的全质量范围检测。
Pressurized capillary electrochromatography (pCEC) has been coupled to an ion trap storage/reflectron time-of-flight mass spectrometer for the analysis of peptide mixtures and protein digests, Taking advantage of the electroosmotic now, high separation efficiency has been achieved in pCEC due to a relatively hat now profile and the use of smaller packing materials, A supplementary pressure was used in these experiments which suppressed bubble formation and also allowed the tuning of the elution of peptides using the electrical field, In this work, a fast separation of a six-peptide mixture has been successfully performed. Using columns only 6 cm long, a tryptic digest of bovine cytochrome c was fully separated in around 14 min by properly tuning the applied voltage and the supplementary pressure, In addition, relatively complex protein digests, such as a tryptic digest of chicken ovalbumin, were analyzed using this pCEC/MS system, and more than 20 peaks were resolved in the total ion current chromatogram within 17 min, The use of an ion trap storage/reflectron time-of-flight mass spectrometer as an on-line detector further increased the resolving power of the pCEC by unambiguously identifying coeluting components, The nonscanning property of the time-of-flight mass analyzer and the ion signal integration capability of the ion trap were successfully combined to provide rapid and sensitive full-mass range detection in these experiments.