Biochemical Mass Spectrometry Laboratory, Centers for Disease Control and Prevention, 4770 Buford Hwy, NE, Mail Stop F-19, Atlanta, GA, 30341, United States.
Clin Chim Acta. 2013 Sep 23;424:191-200. doi: 10.1016/j.cca.2013.06.007. Epub 2013 Jun 21.
Hemoglobinopathies are mutations resulting in abnormal globin chain structure; some have clinically significant outcomes such as anemia or reduced lifespan. Five β-globin mutations are (c.20A>T, p.E6V), (c.19G>A, p. E6K), (c.79G>A, p.E26K), (c.364G>C, p.E121Q), and (c.364G>A, p.E121K), resulting in HbS (sickle-cell hemoglobin), HbC, HbE, HbD-Los Angeles, and HbO-Arab, respectively. One α-globin mutation is (c.[207C>G or 207C>A], p.N68K), resulting in HbG-Philadelphia.
HPLC-ESI-MS/MS analysis of dried-blood spot (DBS) punches from newborns extracted with a trypsin-containing solution provides greater than 90% coverage of α-, β-, and γ-globin amino acid sequences. Because the (c.20A>T, p.E6V), (c.19G>A, p. E6K), (c.79G>A, p.E26K), (c.364G>C, p.E121Q), (c.364G>A, p.E121K), and (c.[207C>G or 207C>A], p.N68K) mutations generate globin peptides with novel amino acid sequences, detecting one of these peptides in DBS extracts is indicative of the presence of a hemoglobinopathy in the newborn.
The method described here can distinguish normal β-globin peptides from the mutant HbS, HbC, HbE, HbD-Los Angeles and HbO-Arab peptides, as well as normal α-globin peptide from the mutant HbG-Philadelphia peptide, allowing the identification of unaffected heterozygotes such as HbAS, and of compound heterozygotes such as HbASG-Philadelphia.
This HPLC-ESI-MS/MS analytical approach provides information that is not available from traditional hemoglobin analyses such as isoelectric focusing and HPLC-UV. It is also capable of determining the amino acid sequence of hemoglobin peptides, potentially allowing the detection of numerous hemoglobinopathies resulting from point mutations.
血红蛋白病是导致珠蛋白链结构异常的突变；有些具有临床显著的结果，如贫血或寿命缩短。五种β-珠蛋白突变分别为(c.20A>T，p.E6V)、(c.19G>A，p.E6K)、(c.79G>A，p.E26K)、(c.364G>C，p.E121Q)和(c.364G>A，p.E121K)，分别导致 HbS(镰状血红蛋白)、HbC、HbE、HbD-洛杉矶和 HbO-阿拉伯。一种α-珠蛋白突变是(c.[207C>G 或 207C>A]，p.N68K)，导致 HbG-费城。
用含胰蛋白酶的溶液提取新生儿干血斑(DBS)斑点进行高效液相色谱-电喷雾串联质谱(LC-ESI-MS/MS)分析，可提供超过 90%的α-、β-和γ-珠蛋白氨基酸序列覆盖。由于(c.20A>T，p.E6V)、(c.19G>A，p.E6K)、(c.79G>A，p.E26K)、(c.364G>C，p.E121Q)、(c.364G>A，p.E121K)和(c.[207C>G 或 207C>A]，p.N68K)突变生成具有新氨基酸序列的珠蛋白肽，在 DBS 提取物中检测到其中一种肽表明新生儿存在血红蛋白病。
本文描述的方法可以区分正常的β-珠蛋白肽与突变的 HbS、HbC、HbE、HbD-洛杉矶和 HbO-阿拉伯肽，以及正常的α-珠蛋白肽与突变的 HbG-费城肽，从而可以鉴定未受影响的杂合子，如 HbAS，以及复合杂合子，如 HbASG-费城。
这种 LC-ESI-MS/MS 分析方法提供了传统血红蛋白分析(如等电聚焦和 LC-UV)无法提供的信息。它还能够确定血红蛋白肽的氨基酸序列，有可能检测到许多由点突变引起的血红蛋白病。
