Krabbe J G, Gao F, Li J, Ahlskog J E, Lingeman H, Niessen W M A, Irth H
Department of Analytical Chemistry and Applied Spectroscopy, Division of Chemistry, Faculty of Sciences, Vrije Universiteit Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
J Chromatogr A. 2006 Oct 20;1130(2):287-95. doi: 10.1016/j.chroma.2006.05.085. Epub 2006 Jul 3.
A ligand-exchange method for the detection and identification of phosphorylated peptides in complex mixtures is presented that is based on the characterization of phosphorylated species by solution-phase interactions with Fe(III) ions and subsequent fluorescence readout. After the separation of the peptides and digest products on a reversed-phase LC column, the flow is split between the two detection systems. One part is directed towards an electrospray mass spectrometer for direct detection and identification of all the peptides present in the sample. The other part of the flow is directed towards a ligand-exchange detection system. This system relies on the specific release of a fluorescent reporter ligand from a Fe(III)-complex in the presence of phosphorylated peptides. To recognize false positive signals due to high-affinity non-phosphorylated high-acidic peptides and other compounds which are known to be a problem in for instance immobilized metal affinity chromatography (IMAC), a second run is performed after incubation of the sample with alkaline phosphatase. A positive signal in this second run indicates a high-affinity non-phosphorylated compound. The method is illustrated using digest from a phosphorylated alpha-casein. Automated switching between MS and MS-MS was performed to obtain additional information about the compounds present in the sample. The linearity of the method was tested in the range of 0.5-80 microM of phosphorylated peptides. A limit of detection (LOD) of 0.5 microM was obtained for a mono-phosphorylated peptide. The interday (n=4) and intraday precision (n=3) expressed as relative standard deviation was better than 10%.
本文介绍了一种用于检测和鉴定复杂混合物中磷酸化肽段的配体交换方法，该方法基于通过与Fe(III)离子的溶液相相互作用对磷酸化物种进行表征并随后进行荧光读数。在反相液相色谱柱上分离肽段和消化产物后，将流动相分流到两个检测系统。一部分流向电喷雾质谱仪，用于直接检测和鉴定样品中存在的所有肽段。流动相的另一部分流向配体交换检测系统。该系统依赖于在磷酸化肽段存在下从Fe(III)络合物中特异性释放荧光报告配体。为了识别由于高亲和力非磷酸化高酸性肽和其他化合物（例如在固定化金属亲和色谱（IMAC）中已知存在问题的化合物）导致的假阳性信号，在样品与碱性磷酸酶孵育后进行第二次运行。第二次运行中的阳性信号表明存在高亲和力非磷酸化化合物。使用磷酸化α-酪蛋白的消化产物对该方法进行了说明。进行了MS和MS-MS之间的自动切换，以获取有关样品中存在的化合物的更多信息。在0.5-80 microM的磷酸化肽段范围内测试了该方法的线性。对于单磷酸化肽段，获得的检测限（LOD）为0.5 microM。以相对标准偏差表示的日间（n = 4）和日内精密度（n = 3）优于10%。
