Falick A M, Shackleton C H, Green B N, Witkowska H E
Department of Pharmaceutical Chemistry, University of California, San Francisco.
Rapid Commun Mass Spectrom. 1990 Oct;4(10):396-400. doi: 10.1002/rcm.1290041010.
A combination of mass spectrometric techniques (electrospray mass spectrometry, liquid secondary-ion mass spectrometry (LSIMS), tandem mass spectrometry) has been used for variant hemoglobin detection and characterization. Electrospray mass spectrometry allowed analysis of mixtures of intact globins giving the molecular weights (accuracy 1-2 Da), and information about relative amounts of globins present, simultaneously. Abnormal hemoglobins detected in this way and by other means (screening, clinical symptoms) were fractionated by C-4 reverse phase high-performance liquid chromatography (HPLC), and the separated globin chains (or the mixture of whole precipitated globin) were digested with trypsin. The tryptic peptides were separated by C-18 reverse phase HPLC and analysed by LSIMS to narrow down the mutation site to a single peptide. In some instances, the molecular weight of a variant peptide was sufficient to determine the mutation uniquely. When molecular weight information alone was insufficient to identify the mutation and its site, the peptide was sequenced by tandem mass spectrometry on a 4-sector instrument. In cases where more than one possible mutation site was present in the peptide and the mutation resulted in a change of only 1 Da in the peptide mass, the resolution and mass measurement accuracy of the 4-sector machine were essential in determining the correct sequence. The practical application of the methodologies presented is illustrated by the identification and analysis of Hb G-San Jose, Hb Willamette and D-Iran.
多种质谱技术（电喷雾质谱、液体二次离子质谱（LSIMS）、串联质谱）已被用于变异血红蛋白的检测和表征。电喷雾质谱可对完整珠蛋白混合物进行分析，同时给出分子量（准确度为1 - 2道尔顿）以及有关存在的珠蛋白相对含量的信息。通过这种方法以及其他手段（筛查、临床症状）检测到的异常血红蛋白，用C - 4反相高效液相色谱（HPLC）进行分离，分离出的珠蛋白链（或整个沉淀珠蛋白的混合物）用胰蛋白酶消化。胰蛋白酶消化后的肽段用C - 18反相HPLC分离，并用LSIMS分析，以将突变位点缩小到单个肽段。在某些情况下，变异肽段的分子量足以唯一确定突变。当仅分子量信息不足以识别突变及其位点时，该肽段在四扇区仪器上通过串联质谱进行测序。在肽段中存在多个可能的突变位点且突变导致肽段质量仅变化1道尔顿的情况下，四扇区仪器的分辨率和质量测量准确度对于确定正确序列至关重要。通过对Hb G - 圣何塞、Hb威拉米特和D - 伊朗血红蛋白的鉴定和分析说明了所介绍方法的实际应用。
