种子是食物蛋白质的主要来源,对人类营养和动物饲养都有影响。因此,种子质量需要在生存能力和食品安全的背景下适当解决。的确,种子的长期和不适当的储存可能导致蛋白质糖基化的增强,这可能会影响它们的质量和寿命。种子蛋白的糖基化可以通过彻底的酸水解和通过液相色谱-质谱(LC-MS)对糖基化加合物N-(羧甲基)赖氨酸(CML)进行定量来探测。这种方法,然而,不允许分析热和化学不稳定的糖基化加合物,像乙二醛-,甲基乙二醛和3-脱氧葡萄糖酮衍生的氢咪唑酮。尽管在这种情况下,酶促水解可能是一个很好的解决方案,它需要水性条件,这不能确保种子蛋白分离物的重建。正因为如此,到目前为止,尚未对种子晚期糖基化终产物(AGEs)的完整特征进行表征。因此,在这里我们提出的方法,在十二烷基硫酸钠(SDS)存在下,种子蛋白的定量溶解及其定量酶促水解,然后通过反相固相萃取(RP-SPE)去除SDS。以甲基乙二醛衍生的氢咪唑酮1(MG-H1)为例,我们证明了该方法用于可靠和灵敏的基于LC-MS的化学不稳定AGEs定量的适用性及其与生物测定的兼容性。
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 Nɛ-(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.