Thakkar Amit, Wavreille Anne-Sophie, Pei Dehua
Department of Chemistry and Ohio State Biochemistry Program, Ohio State University, 100 West 18th Avenue, Columbus, Ohio 43210, USA.
Anal Chem. 2006 Aug 15;78(16):5935-9. doi: 10.1021/ac0607414.
An improved method for the rapid sequence determination of biologically active peptides selected from one-bead-one-peptide combinatorial libraries has been developed. In this method, beads carrying unique peptide sequences were subjected to multiple cycles of partial Edman degradation (PED) by the treatment with a 15-30:1 mixture of phenyl isothiocyanate and N-(9-fluorenylmethoxycarbonyloxy)succinimide (Fmoc-OSU), to generate a series of sequence-specific truncation products (a peptide ladder) for each resin-bound peptide. Following PED, the Fmoc group was removed from the N-terminus and any reacted side chains by piperidine treatment. The sequence of the full-length peptide on each bead was then determined by matrix-assisted laser desorption ionization mass spectrometry. The use of Fmoc-OSU as a traceless capping agent resulted in cleaner MS spectra and improved reliability for sequence assignment. This rapid, sensitive, and inexpensive sequencing method should further expand the utility of combinatorial peptide libraries in biomedical research.
已开发出一种改进方法，用于从单珠单肽组合文库中快速测定生物活性肽的序列。在该方法中，携带独特肽序列的珠子通过用异硫氰酸苯酯和N-(9-芴甲氧羰基氧基)琥珀酰亚胺（Fmoc-OSU）的15-30:1混合物处理进行多轮部分埃德曼降解（PED），为每个树脂结合肽生成一系列序列特异性截短产物（肽梯）。PED之后，通过哌啶处理从N端和任何反应的侧链上去除Fmoc基团。然后通过基质辅助激光解吸电离质谱法测定每个珠子上全长肽的序列。使用Fmoc-OSU作为无痕封端剂可产生更清晰的质谱图并提高序列分配的可靠性。这种快速、灵敏且廉价的测序方法应会进一步扩大组合肽文库在生物医学研究中的应用。
