Schopfer Lawrence M, Champion Matthew M, Tamblyn Nate, Thompson Charles M, Lockridge Oksana
University of Nebraska Medical Center, Eppley Institute, Omaha, NE 68198, USA.
Anal Biochem. 2005 Oct 1;345(1):122-32. doi: 10.1016/j.ab.2005.07.016.
A mass spectrometry-based method was developed for selective detection of FP-biotinylated peptides in complex mixtures. Mixtures of peptides, at the low-picomole level, were analyzed by liquid chromatography and positive ion, nanospray, triple quadrupole, linear ion trap mass spectrometry. Peptides were fragmented by collision-activated dissociation in the mass spectrometer. The free FP-biotin and peptides containing FP-biotinylated serine or FP-biotinylated tyrosine yielded characteristic fragment ions at 227, 312, and 329 m/z. FP-biotinylated serine yielded an additional characteristic fragment ion at 591 m/z. Chromatographic peaks containing FP-biotinylated peptides were indicated by these diagnostic ions. Data illustrating the selectivity of the approach are presented for tryptic digests of FP-biotinylated trypsin and FP-biotinylated serum albumin. A 16-residue peptide from bovine trypsin was biotinylated on the active site serine. A 3-residue peptide from bovine albumin, YTR, was biotinylated on Tyr410. This latter result confirms that the organophosphorus binding site of albumin is a tyrosine. This method can be used to search for new biomarkers of organophosphorus agent exposure.
开发了一种基于质谱的方法，用于在复杂混合物中选择性检测FP-生物素化肽段。低皮摩尔水平的肽混合物通过液相色谱和正离子、纳喷、三重四极杆、线性离子阱质谱进行分析。肽段在质谱仪中通过碰撞诱导解离进行碎裂。游离的FP-生物素以及含有FP-生物素化丝氨酸或FP-生物素化酪氨酸的肽段在质荷比为227、312和329处产生特征性碎片离子。FP-生物素化丝氨酸在质荷比为591处还产生一个额外的特征性碎片离子。含有FP-生物素化肽段的色谱峰由这些诊断离子指示。给出了FP-生物素化胰蛋白酶和FP-生物素化血清白蛋白的胰蛋白酶消化产物的数据，以说明该方法的选择性。牛胰蛋白酶的一个16个残基的肽段在活性位点丝氨酸上进行了生物素化。牛白蛋白的一个3个残基的肽段YTR在Tyr410上进行了生物素化。后一结果证实白蛋白的有机磷结合位点是酪氨酸。该方法可用于寻找有机磷剂暴露的新生物标志物。
