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与扇形磁质谱联用。它允许在HPLC/ESI-MS中进行灵敏和准确的质量测量。因此，混合物样品中涉及的所有组分，例如蛋白质的酶消化物，可以在通过毛细管柱（300 mm直径）分离后有效地测量。蛋白质混合物和蛋白水解消化物的分析表明，毛细管HPLC和磁扇形质谱仪的组合能够在皮摩尔量的质量测定中获得优于0.01%的准确度。它可以成功地应用于确定的阅读框架的DNA衍生的蛋白质序列和位点和类型的修饰存在于重组或天然蛋白质。设计了一种与毛细管HPLC联用的微量印迹装置，用于收集从毛细管柱上洗脱到聚偏氟乙烯膜上的样品溶液，该装置能以高回收率（90%以上）分离毛细管柱分离的各组分，可方便地进行氨基酸组成或序列分析。毛细管HPLC/ESI-MS和微量印迹系统的结合将为仅在有限数量下可获得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.