Department of Chemistry and Biochemistry, National Chung Cheng University, 168 University Road, Ming-Hsiung, Chia-Yi 62142, Taiwan.
J Food Drug Anal. 2019 Apr;27(2):526-530. doi: 10.1016/j.jfda.2018.10.003. Epub 2018 Oct 27.
Blood sampling by the dried blood spot (DBS) technique has become commonly applied in newborn screening. It is often used for analysis of small molecules, such as metabolites. Recently, DBS sampling has been applied for quantification of post-translational protein modifications. Glyoxal and methylglyoxal are two simple oxoaldehydes released from glycated proteins in the Maillard reaction. They are widely distributed in the environment (e.g. cigarette smoke) and found in foods and beverages. Glyoxal and methylglyoxal are shown to react with biomolecules including DNA and proteins. In this laboratory, we previously identified the sites of modification by these two oxoaldehydes in human hemoglobin and found that the extents of modification at certain sites of lysine and arginine residues are significantly higher in type 2 diabetes mellitus patients than in nondiabetic individuals. In this study, we examine the stability of these modifications of hemoglobin stored on DBS cards at room temperature or 4 °C in the ambient air. After hemoglobin was extracted from the DBS cards, it was digested by trypsin and analyzed by nanoflow liquid chromatography coupled with nanospray ionization tandem mass spectrometry. The results show that the extents of all these PTMs are stable within 14 and 21 days when stored on DBS at room temperature and at 4 °C, respectively. Extraction of globin from DBS cards is mostly advantageous for hemolytic blood samples. This assay is sensitive as only a quarter of a DBS card containing ca. 12 μL of blood is required. Thus, it is practically useful to measure the extents of glyoxal- and methylglyoxal-induced hemoglobin modifications from DBS cards.
干血斑（DBS）技术采血已广泛应用于新生儿筛查。它通常用于分析小分子，如代谢物。最近，DBS 采样已应用于翻译后蛋白质修饰的定量分析。乙二醛和甲基乙二醛是美拉德反应中糖化蛋白质释放的两种简单的氧代醛。它们广泛分布于环境（如香烟烟雾）中，并存在于食品和饮料中。乙二醛和甲基乙二醛与包括 DNA 和蛋白质在内的生物分子反应。在本实验室中，我们先前在人血红蛋白中鉴定了这两种氧代醛修饰的位点，并发现某些赖氨酸和精氨酸残基位点的修饰程度在 2 型糖尿病患者中明显高于非糖尿病个体。在这项研究中，我们检查了在室温或 4°C 下在环境空气中保存在 DBS 卡上的血红蛋白修饰的稳定性。从 DBS 卡中提取血红蛋白后，用胰蛋白酶消化，并用纳流液相色谱与纳喷雾电离串联质谱分析。结果表明，在室温下保存在 DBS 卡上时，所有这些 PTM 的程度在 14 天和 21 天内稳定，在 4°C 下保存在 DBS 卡上时，在 14 天和 21 天内稳定。从 DBS 卡中提取球蛋白对溶血血样大多有利。该测定法灵敏，因为仅需四分之一的 DBS 卡（含约 12μL 的血液）即可。因此，从 DBS 卡测量乙二醛和甲基乙二醛诱导的血红蛋白修饰的程度实际上是有用的。
