Manchester Institute of Biotechnology , University of Manchester , 131 Princess Street , Manchester M1 7DN , United Kingdom.
Department of Chemistry , University of Manchester , Oxford Road , Manchester M13 9PL , United Kingdom.
Anal Chem. 2019 Sep 17;91(18):11502-11506. doi: 10.1021/acs.analchem.9b03319. Epub 2019 Aug 29.
A convenient approach is proposed for the quantitation of elemental cofactors in proteins and the determination of metal/protein stoichiometry, on the basis of energy dispersive X-ray fluorescence spectroscopy (EDXRF). The analysis of proteins containing the metals Cu, Fe, Zn, and Ca and also the nonmetallic element P is shown as a demonstration of the generality of the method. In general, the reported method gives limit of detection (LOD) and limit of quantification (LOQ) values in the low ppm range and requires only a few microliters of protein sample at micromolar concentrations. Moreover, sample preparation does not require any digestion steps before the analysis. The expected metal/protein stoichiometry was observed for each protein analyzed, highlighting the precision and accuracy of the method in all the tested cases. Furthermore, it is shown that the method is compatible with multimeric proteins and those with post-translational modifications such as glycosylation.
提出了一种基于能量色散 X 射线荧光光谱（EDXRF）的简便方法，用于定量蛋白质中的元素辅因子和测定金属/蛋白质化学计量比。该方法展示了分析含铜、铁、锌、钙金属和非金属元素磷的蛋白质的通用性。通常，所报道的方法在低 ppm 范围内给出检测限（LOD）和定量限（LOQ）值，并且仅需要几微升微摩尔浓度的蛋白质样品。此外，样品制备在分析之前不需要任何消化步骤。对于分析的每种蛋白质，都观察到了预期的金属/蛋白质化学计量比，突出了该方法在所有测试情况下的精确性和准确性。此外，还表明该方法与多聚体蛋白质以及那些经过翻译后修饰（如糖基化）的蛋白质兼容。
