Fast photochemical oxidation of proteins (FPOP) may be used to characterize changes in protein structure by measuring differences in the apparent rate of peptide oxidation by hydroxyl radicals. The variability between replicates is high for some peptides and limits the statistical power of the technique, even using modern methods controlling variability in radical dose and quenching. Currently, the root cause of this variability has not been systematically explored, and it is unknown if the major source(s) of variability are structural heterogeneity in samples, remaining irreproducibility in FPOP oxidation, or errors in LC-MS quantification of oxidation. In this work, we demonstrate that coefficient of variation of FPOP measurements varies widely at low peptide signal intensity, but stabilizes to ≈ 0.13 at higher peptide signal intensity. We dramatically reduced FPOP variability by increasing the total sample loaded onto the LC column, indicating that the major source of variability in FPOP measurements is the difficulties in quantifying oxidation at low peptide signal intensities. This simple method greatly increases the sensitivity of FPOP structural comparisons, an important step in applying the technique to study subtle conformational changes and protein-ligand interactions.

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蛋白质的快速光化学氧化（FPOP）可用于通过测量羟基自由基氧化肽的表观速率的差异来表征蛋白质结构的变化。对于某些多肽，重复之间的变异性很高，即使使用控制自由基剂量和猝灭变异性的现代方法，也限制了该技术的统计能力。目前，尚未对该变异性的根本原因进行系统地探讨，并且不确定性的主要来源是否是样品中的结构异质性，FPOP氧化中仍存在不可再现性或LC-MS氧化定量中的错误。在这项工作中，我们证明了FPOP测量的变异系数在低肽信号强度时变化很大，但在高肽信号强度时稳定到≈0.13。我们通过增加上样至LC色谱柱的总样品量大大降低了FPOP的可变性，这表明FPOP测量中可变性的主要来源是难以在低肽信号强度下定量氧化。这种简单的方法大大提高了FPOP结构比较的灵敏度，这是应用该技术研究微妙的构象变化和蛋白质-配体相互作用的重要一步。

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