Witkowska H E, Bitsch F, Shackleton C H
Mass Spectrometry Facility, Children's Hospital Oakland Research Institute, CA 94609.
Hemoglobin. 1993 Jun;17(3):227-42. doi: 10.3109/03630269308998897.
Microscale analysis of a variant hemoglobin (Hb) has been achieved by combination of high performance liquid chromatography (HPLC) and electrospray mass spectrometry (ESMS) and the method should be almost universally applicable. We have eliminated preparative scale HPLC of globin chains and semi-preparative HPLC of proteolytic digests which had been used prior to mass spectrometry. Use of microbore HPLC columns reduced the time required for analysis substantially and solvent usage by 100x. Molecular masses of intact globins and masses and sequence information of tryptic peptides could be obtained without collecting and separately analyzing chromatographic fractions. As an example of the use of these methods, we report the characterization of an unknown hemoglobinopathy case that was finally authenticated as Hb P-Galveston [beta 117(G19)His-->Arg], using the following sequence of analyses: 1) ESMS of complete hemolysate, 2) analytical HPLC of globin chains, 3) combined microbore HPLC/ESMS of globin chains to determine their molecular masses, 4) cysteine derivatization and tryptic digestion of mixture of all globins, followed by microbore separation of the peptides, molecular mass determination, and generation of fragmentation patterns allowing confirmation of amino acid sequences. This four-part strategy should allow characterization of almost all variant Hbs. Exceptions would be mutations in regions of globin chains which give rise to small (< four residues) tryptic peptides, either normal or produced by addition of new tryptic sites and mutations that introduce only minute difference in molecular weight (MW) of tryptic peptides. Since only 10% of each separated peptides is mass analyzed, 90% is available for collection and further structural identification (e.g. by tandem MS or Edman sequencing) if the identity is still in doubt.
通过高效液相色谱（HPLC）和电喷雾质谱（ESMS）联用实现了对一种变异血红蛋白（Hb）的微量分析，该方法几乎普遍适用。我们省去了在质谱分析之前使用的珠蛋白链的制备规模HPLC和蛋白水解消化产物的半制备HPLC。使用微径HPLC柱大大减少了分析所需时间，溶剂用量减少了100倍。无需收集和单独分析色谱馏分，就可以获得完整珠蛋白的分子量以及胰蛋白酶肽段的分子量和序列信息。作为这些方法应用的一个例子，我们报告了一例未知血红蛋白病病例的特征分析，该病例最终被鉴定为Hb P - 加尔维斯顿[β117（G19）组氨酸→精氨酸]，采用了以下分析顺序：1）完整溶血产物的ESMS；2）珠蛋白链的分析型HPLC；3）珠蛋白链的微径HPLC/ESMS联用，以确定其分子量；4）所有珠蛋白混合物的半胱氨酸衍生化和胰蛋白酶消化，随后对肽段进行微径分离、分子量测定，并生成裂解模式以确认氨基酸序列。这一四部分策略应能对几乎所有变异Hb进行特征分析。例外情况是珠蛋白链区域的突变，这些突变会产生小的（<4个残基）胰蛋白酶肽段，无论是正常的还是由新的胰蛋白酶位点添加产生以及那些仅在胰蛋白酶肽段分子量（MW）上引入微小差异的突变。由于每次分离的肽段只有10%进行了质量分析，如果身份仍有疑问，90%可用于收集和进一步的结构鉴定（例如通过串联质谱或埃德曼测序）。
