Department of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry, 06120 Halle, Germany.
Department of Biochemistry, St. Petersburg State University, 199004 St. Petersburg, Russia.
Int J Mol Sci. 2019 Jul 26;20(15):3659. doi: 10.3390/ijms20153659.
Seeds represent the major source of food protein, impacting on both human nutrition and animal feeding. Therefore, seed quality needs to be appropriately addressed in the context of viability and food safety. Indeed, long-term and inappropriate storage of seeds might result in enhancement of protein glycation, which might affect their quality and longevity. Glycation of seed proteins can be probed by exhaustive acid hydrolysis and quantification of the glycation adduct -(carboxymethyl)lysine (CML) by liquid chromatography-mass spectrometry (LC-MS). This approach, however, does not allow analysis of thermally and chemically labile glycation adducts, like glyoxal-, methylglyoxal- and 3-deoxyglucosone-derived hydroimidazolones. Although enzymatic hydrolysis might be a good solution in this context, it requires aqueous conditions, which cannot ensure reconstitution of seed protein isolates. Because of this, the complete profiles of seed advanced glycation end products (AGEs) are not characterized so far. Therefore, here we propose the approach, giving access to quantitative solubilization of seed proteins in presence of sodium dodecyl sulfate (SDS) and their quantitative enzymatic hydrolysis prior to removal of SDS by reversed phase solid phase extraction (RP-SPE). Using methylglyoxal-derived hydroimidazolone 1 (MG-H1) as a case example, we demonstrate the applicability of this method for reliable and sensitive LC-MS-based quantification of chemically labile AGEs and its compatibility with bioassays.
种子是蛋白质的主要食物来源，对人类营养和动物饲养都有影响。因此，在考虑种子活力和食品安全的情况下，需要适当关注种子质量。事实上，种子如果长期储存不当，可能会导致蛋白质糖基化增强，从而影响其质量和保质期。种子蛋白的糖基化可以通过彻底的酸水解和液相色谱-质谱（LC-MS）对糖基化加合物-(羧甲基)赖氨酸（CML）的定量来探测。然而，这种方法不能分析热不稳定和化学不稳定的糖基化加合物，如乙二醛、甲基乙二醛和 3-脱氧葡萄糖酮衍生的羟咪唑啉酮。尽管在这种情况下酶解可能是一个很好的解决方案，但它需要水相条件，这不能保证种子蛋白分离物的重组。正因为如此，目前还没有对种子晚期糖基化终产物（AGEs）的完整图谱进行特征描述。因此，在这里我们提出了一种方法，该方法可以在十二烷基硫酸钠（SDS）存在的情况下定量溶解种子蛋白，并在通过反相固相萃取（RP-SPE）去除 SDS 之前对其进行定量酶解。以甲基乙二醛衍生的羟咪唑啉酮 1（MG-H1）为例，我们证明了该方法在基于 LC-MS 的可靠和敏感化学不稳定 AGE 定量方面的适用性，以及其与生物测定的兼容性。
