Pathology Department, University of Otago, Christchurch, New Zealand; Canterbury Health Laboratories, Christchurch, New Zealand.
Canterbury Health Laboratories, Christchurch, New Zealand.
Clin Chim Acta. 2015 Feb 2;440:40-3. doi: 10.1016/j.cca.2014.11.003. Epub 2014 Nov 7.
Serum protein electrophoresis occasionally reveals multiple albumin bands referred to as bis- or alloalbuminaemia, and whilst the condition can be inherited it may also be acquired.
We present a new high resolution approach to the investigation of qualitative changes in albumin structure. The on-line reverse phase time-of-flight mass spectrometry procedure (TOF MS) elaborated here requires <0.2 μl of plasma and takes ~10 min to perform. Two plasma samples with classical bisalbuminaemia were used to verify the efficacy of the procedure for detecting genetic variants. When a novel mutation was detected, mass mapping of native unreduced albumin was used to pinpoint its location and inform targeted DNA sequencing of the albumin gene.
Normal serum albumin showed its expected major isoform at 66,439 Da and the electrophoretic variants showed co-equal expression of additional components at -14 and +744 Da respectively. Surprisingly, one of the supposed controls showed paired albumin peaks at 66,439 and 66,469 Da and this 30 Da increase in mass was localised to between Arg(114) and Arg(197) and confirmed as being due to a novel 191Ala→Thr (+30 Da) substitution through DNA sequencing.
The electrospray TOF MS approach developed here provides a rapid, sensitive and extremely precise method of revealing minute changes in albumin primary structure.
血清蛋白电泳偶尔会显示出多个白蛋白条带，称为双白蛋白或异白蛋白血症，这种情况既可以遗传，也可以后天获得。
我们提出了一种新的高分辨率方法来研究白蛋白结构的定性变化。这里详细阐述的在线反相飞行时间质谱（TOF MS）程序需要<0.2 μl 的血浆，并且大约需要 10 分钟才能完成。使用两种具有经典双白蛋白血症的血浆样品来验证该程序检测遗传变异的效果。当检测到新的突变时，使用天然未还原白蛋白的质量映射来确定其位置，并为白蛋白基因的靶向 DNA 测序提供信息。
正常血清白蛋白在 66,439 Da 处显示其预期的主要同工型，电泳变异体分别在-14 和+744 Da 处显示出相等的额外成分表达。令人惊讶的是，其中一个所谓的对照在 66,439 和 66,469 Da 处显示出配对的白蛋白峰，质量增加 30 Da，局部定位于 Arg(114)和 Arg(197)之间，并通过 DNA 测序证实是由于 novel 191Ala→Thr (+30 Da)取代。
这里开发的电喷雾 TOF MS 方法提供了一种快速、敏感和极其精确的方法来揭示白蛋白一级结构的微小变化。
