Research Reactor Institute, Kyoto University Kumatori-cho, Sennan-gun, Osaka 590-0494, Japan.
Department of Chemistry, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
Biochim Biophys Acta Proteins Proteom. 2018 Jul;1866(7):767-774. doi: 10.1016/j.bbapap.2018.04.002. Epub 2018 Apr 11.
Many post-translational modifications such as oxidation, deamidation and isomerization of amino acid residues occur in lens proteins with aging. One such modification, isomerization of aspartate in lens α-crystallin, has been well studied by amino acid enantiomer analysis and LC-MS/MS. LC-MS/MS can quickly and easily identify D- and L-amino acid-containing peptides without purification of lens protein mixtures. However, this method has a weak point in that isomeric peptides of major components are detected predominantly, while those from minor proteins such as β- and γ-crystallins have not been fully determined. Therefore, the isomerization of amino acid residues in β- and γ-crystallin families has been little studied. To solve those problems and detect the isomerization of Asp residues in lens βB2-crystallin, the main component of the β-crystallin family, here we have developed steps for sample fractionation before d/l analysis based on either LC-MS/MS or amino acid derivatization to diastereoisomers followed by RP-HPLC. To capture a small amount of peptide, a multiple reaction monitoring (MRM) method based on quadrupole MS/MS (Q-MS) was applied to the water-soluble fraction of whole lens. The d/l analysis based on both LC-MS/MS and diastereoisomer formation showed the presence of multiple isomerization sites, including Asp4, Asp83, Asp92 and Asp192, in βB2-crystallin in aged lens. These isomerization sites were confirmed to exist in an age-dependent manner by Q-MS. Synthetic peptides of βB2-crystallin containing different isomers of Asp showed differential elution profiles during RP-HPLC, indicating differences in the local structure or hydrophobicity of Asp-isomer-containing peptides. These results suggest that the isomerization sites are distributed on exposed regions of βB2-crystallin and thus likely to have an impact on crystallin subunit-subunit interactions, induce abnormal crystallin aggregation, and contribute to senile cataract formation in aged lens.
随着年龄的增长，晶状体蛋白会发生许多翻译后修饰，如氨基酸残基的氧化、脱酰胺和异构化。其中一种修饰是晶状体α-晶体蛋白中天冬氨酸的异构化，已通过氨基酸对映体分析和 LC-MS/MS 进行了很好的研究。LC-MS/MS 可以快速轻松地鉴定不含透镜蛋白混合物的 D-和 L-氨基酸肽，而无需对其进行纯化。然而，这种方法有一个弱点，即主要成分的异构肽主要被检测到，而β-和γ-晶体等次要蛋白质的异构肽则尚未完全确定。因此，β-和γ-晶体家族中氨基酸残基的异构化研究甚少。为了解决这些问题并检测晶状体βB2-晶体素中氨基酸残基的异构化，β-晶体素家族的主要成分，我们在此开发了基于 LC-MS/MS 或氨基酸衍生化的样品分步分离方法，以形成非对映异构体，然后进行反相高效液相色谱（RP-HPLC）。为了捕获少量肽，我们在全晶状体的水溶性部分应用了基于四极杆 MS/MS（Q-MS）的多重反应监测（MRM）方法。基于 LC-MS/MS 和非对映异构体形成的 d/l 分析表明，在衰老的晶状体中，βB2-晶体素存在多个异构化位点，包括 Asp4、Asp83、Asp92 和 Asp192。通过 Q-MS 证实这些异构化位点以年龄依赖的方式存在。含有不同 Asp 异构体的βB2-晶体素合成肽在 RP-HPLC 中显示出不同的洗脱曲线，表明含 Asp-异构体的肽的局部结构或疏水性存在差异。这些结果表明，异构化位点分布在βB2-晶体素的暴露区域，因此可能对晶体素亚基-亚基相互作用产生影响，诱导异常晶体素聚集，并导致衰老晶状体中白内障的形成。
