Thoma R S, Smith J S, Sandoval W, Leone J W, Hunziker P, Hampton B, Linse K D, Denslow N D
Monsanto Co., St. Louis, MO 63167, USA.
J Biomol Tech. 2009 Sep;20(4):216-25.
The Edman Sequence Research Group (ESRG) of the Association of Biomolecular Resource designs and executes interlaboratory studies investigating the use of automated Edman degradation for protein and peptide analysis. In 2008, the ESRG enlisted the help of core sequencing facilities to investigate the effects of a repeating amino acid tag at the N-terminus of a protein. Commonly, to facilitate protein purification, an affinity tag containing a polyhistidine sequence is conjugated to the N-terminus of the protein. After expression, polyhistidine-tagged protein is readily purified via chelation with an immobilized metal affinity resin. The addition of the polyhistidine tag presents unique challenges for the determination of protein identity using Edman degradation chemistry. Participating laboratories were asked to sequence one protein engineered in three configurations: with an N-terminal polyhistidine tag; with an N-terminal polyalanine tag; or with no tag. Study participants were asked to return a data file containing the uncorrected amino acid picomole yields for the first 17 cycles. Initial and repetitive yield (R.Y.) information and the amount of lag were evaluated. Information about instrumentation and sample treatment was also collected as part of the study. For this study, the majority of participating laboratories successfully called the amino acid sequence for 17 cycles for all three test proteins. In general, laboratories found it more difficult to call the sequence containing the polyhistidine tag. Lag was observed earlier and more consistently with the polyhistidine-tagged protein than the polyalanine-tagged protein. Histidine yields were significantly less than the alanine yields in the tag portion of each analysis. The polyhistidine and polyalanine protein-R.Y. calculations were found to be equivalent. These calculations showed that the nontagged portion from each protein was equivalent. The terminal histidines from the tagged portion of the protein were demonstrated to be responsible for the high lag during N-terminal sequence analysis.
生物分子资源协会的埃德曼序列研究小组（ESRG）设计并开展了实验室间研究，以调查自动埃德曼降解法在蛋白质和肽分析中的应用。2008年，ESRG寻求核心测序设施的帮助，以研究蛋白质N端重复氨基酸标签的影响。通常，为便于蛋白质纯化，会将含有多组氨酸序列的亲和标签与蛋白质的N端偶联。表达后，带有多组氨酸标签的蛋白质可通过与固定化金属亲和树脂螯合而轻松纯化。多组氨酸标签的添加给使用埃德曼降解化学法确定蛋白质身份带来了独特挑战。参与研究的实验室被要求对一种以三种构型设计的蛋白质进行测序：带有N端多组氨酸标签；带有N端聚丙氨酸标签；或无标签。要求研究参与者返回一个数据文件，其中包含前17个循环未经校正的氨基酸皮摩尔产量。评估了初始产量和重复产量（R.Y.）信息以及滞后量。作为研究的一部分，还收集了有关仪器和样品处理的信息。在这项研究中，大多数参与实验室成功地对所有三种测试蛋白质的1个循环的氨基酸序列进行了测定。一般来说，实验室发现对含有多组氨酸标签的序列进行测定更困难。与带有聚丙氨酸标签的蛋白质相比，带有多组氨酸标签的蛋白质更早且更一致地观察到滞后现象。在每次分析的标签部分，组氨酸产量明显低于丙氨酸产量。发现多组氨酸和聚丙氨酸蛋白质的R.Y.计算结果相当。这些计算表明每种蛋白质的无标签部分相当。蛋白质标签部分的末端组氨酸被证明是N端序列分析中高滞后现象的原因。
