Capillary HPLC/electrospray ionization mass spectrometry for structural analysis of micro-quantities of proteins

通过改进电喷雾离子源，将毛细管高效液相色谱（HPLC）与磁扇形质谱仪相结合。它可以在HPLC/ESI-MS中实现灵敏、准确的质量测量。因此，在毛细管柱（直径300毫米）分离后，可以有效地测量混合样品中涉及的所有组分，例如酶解蛋白质。对蛋白质混合物和蛋白质水解消化液的分析表明，毛细管高效液相色谱和磁扇形质谱联用测定皮摩尔质量的准确度优于0.01%。它可以成功地应用于dna衍生蛋白序列的阅读框以及重组蛋白或天然蛋白中存在的修饰位点和类型的测定。微印迹仪被设计成与毛细管高效液相色谱相结合，用于收集从毛细管柱洗脱到聚偏二氟乙烯膜上的小体积样品溶液，并被证明可以对毛细管柱分离的每种成分进行分选，回收率高（超过90%），可以很容易地进行氨基酸组成或序列分析。毛细管高效液相色谱/ESI-MS和微量印迹系统的结合将为数量有限的肽和蛋白质的微量分析提供一种很好的方法。

Capillary high-performance liquid chromatography (HPLC) has been combined with a magnetic sector mass spectrometer by improving the electrospray ion source. It allows the sensitive and accurate mass measurement in the HPLC/ESI-MS.As a result, all the components involved in a mixture sample such as an enzymatic digest of a protein could be efficiently measured following separation by a capillary column (300 mm diameter). Analyzes of a protein mixture and proteolytic digest indicate that the combination of a capillary HPLC and magnetic sector mass spectrometer is capable of attaining better than 0.01 % accuracy in mass determination on picomole quantities. It could successfully be applied to determination of the reading frames of DNA-derived protein sequence and both sites and types of modifications present in recombinant or natural proteins. A micro-blotting apparatus, which had been designed to be coupled with a capillary HPLC for collecting small volumes of sample solution eluted from a capillary column onto a polyvinylidene fluoride membrane, was demonstrated to fractionate each component separated by a capillary column with the high recovery (more than 90 %), which could readily be subjected to amino acid composition or sequence analyzes. The combination of a capillary HPLC/ESI-MS and micro-blotting system would go a good way for micro-analysis of peptides and proteins that are available only in limited quantities.