Green Brian N, Williams Jonathan P
Waters Corporation, Simonsway, Manchester, United Kingdom.
J Am Soc Mass Spectrom. 2007 Aug;18(8):1493-8. doi: 10.1016/j.jasms.2007.05.005. Epub 2007 May 17.
Many human hemoglobin variants occur in heterozygotes; that is, the variant and normal hemoglobins are present in the same sample. In a procedure for rapidly identifying such variants by mass spectrometry, mutations that increase the mass by 1 Da require a special approach. One of the steps in this procedure involves digesting the denatured hemoglobin with trypsin and analyzing the resulting peptide mixture by mass spectrometry to identify the mutant peptide. Generally the mutant peptide ion can then be selected as the precursor and sequenced by tandem mass spectrometry to identify or confirm the mutation. However, with heterozygotes in which the mass of the variant is 1 Da higher than normal, the first isotope of the mutant peptide occurs at essentially the same mass as the second isotope of the normal peptide, precluding analysis of the mutant peptide on its own. Product ions from the second isotope of a peptide are doublets, 1 Da apart. The way in which the relative abundance of the components in these doublets varies with the elemental composition of the product ions was predicted from the isotopic abundance of the elements and agreed well with experimental data. These results were applied to the identification of a variant that increases the mass by 1 Da in a heterozygote-that is, beta 131 Gln-->Glu, hemoglobin Camden.
许多人类血红蛋白变体以杂合子形式存在；也就是说，变体血红蛋白和正常血红蛋白存在于同一样本中。在一种通过质谱快速鉴定此类变体的方法中，质量增加1道尔顿（Da）的突变需要一种特殊方法。该方法的步骤之一包括用胰蛋白酶消化变性血红蛋白，并通过质谱分析所得的肽混合物以鉴定突变肽。通常，然后可以选择突变肽离子作为前体，并通过串联质谱进行测序以鉴定或确认突变。然而，对于变体质量比正常质量高1 Da的杂合子，突变肽的第一个同位素出现在与正常肽的第二个同位素基本相同的质量处，从而无法单独分析突变肽。肽的第二个同位素产生的产物离子是相隔1 Da的双峰。根据元素的同位素丰度预测了这些双峰中各组分的相对丰度随产物离子元素组成的变化方式，并且与实验数据吻合良好。这些结果被应用于鉴定杂合子中质量增加1 Da的一种变体，即β131谷氨酰胺→谷氨酸，血红蛋白卡姆登。
