Elliott J I, Stone K L, Williams K R
W.M. Keck Foundation Biotechnology Resource Laboratory, Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06510.
Anal Biochem. 1993 May 15;211(1):94-101. doi: 10.1006/abio.1993.1238.
A sequenceable internal standard has been made that can be added to samples destined for automated amino acid sequencing and that provides a means for continuously monitoring instrument performance. The 41-residue standard peptide is composed of two unnatural amino acids, norleucine and succinyl-lysine, and has the structure [norleucine-(succinyl-lysine)4]8-norleucine. The phenylthiohydantoin derivatives of the latter two amino acids are well separated via reversed-phase HPLC from the phenylthiohydantoin derivatives of the 20 naturally occurring amino acids. In addition, the standard peptide can be readily synthesized and it has excellent solubility, stability, and sequencing characteristics. The placement of norleucine at every fifth residue in this peptide permits accurate repetitive yield and carryover calculations following runs that are as short as six cycles. Use of this internal standard permits the early detection of suboptimal instrument performance and is especially helpful in differentiating between two common reasons (blocked NH2-terminus versus an instrument problem) for the failure of an automated protein sequencer to provide an NH2-terminal sequence.
已制备出一种可测序的内标，它可添加到用于自动氨基酸测序的样品中，并提供一种持续监测仪器性能的方法。这种由41个残基组成的标准肽由两种非天然氨基酸——正亮氨酸和琥珀酰赖氨酸构成，其结构为[正亮氨酸-(琥珀酰赖氨酸)4]8-正亮氨酸。后两种氨基酸的苯硫代乙内酰脲衍生物可通过反相高效液相色谱法与20种天然存在氨基酸的苯硫代乙内酰脲衍生物很好地分离。此外，该标准肽易于合成，具有出色的溶解性、稳定性和测序特性。在该肽中每隔五个残基放置一个正亮氨酸，使得在短至六个循环的运行后，能够准确进行重复产率和残留量计算。使用这种内标可以早期检测到仪器性能欠佳的情况，尤其有助于区分自动蛋白质测序仪无法提供氨基末端序列的两个常见原因（氨基末端封闭与仪器问题）。
