Juskowiak Gary L, McGee Christopher J, Greaves John, Van Vranken David L
Department of Chemistry, 1102 Natural Sciences 2, University of California, Irvine, California 92697-2025, USA.
J Comb Chem. 2008 Sep-Oct;10(5):726-31. doi: 10.1021/cc800087y. Epub 2008 Jul 26.
Cysteine-rich peptides are valued as tags for biarsenical fluorophores and as environmentally important reagents for binding toxic heavy metals. Due to the inherent difficulties created by cysteine, the power of one-bead one-compound (OBOC) libraries has never been applied to the discovery of short cysteine-rich peptides. We have developed the first method for the synthesis, screening, and sequencing of cysteine-rich OBOC peptide libraries. First, we synthesized a heavily biased cysteine-rich OBOC library, incorporating 50% cysteine at each position (Ac-X8-KM-TentaGel). Then, we developed conditions for cysteine alkylation, cyanogen bromide cleavage, and direct MS/MS sequencing of that library at the single bead level. The sequencing efficiency of this library was comparable to a traditional cysteine-free library. To validate screening of cysteine-rich OBOC libraries, we reacted a library with the biarsenical FlAsH and identified beads bearing the known biarsenical-binding motif (CCXXCC). These results enable OBOC libraries to be used in high-throughput discovery of cysteine-rich peptides for protein tagging, environmental remediation of metal contaminants, or cysteine-rich pharmaceuticals.
富含半胱氨酸的肽作为双砷荧光团的标签以及用于结合有毒重金属的对环境重要的试剂而受到重视。由于半胱氨酸带来的固有困难，单珠单化合物（OBOC）文库的强大功能从未应用于富含半胱氨酸的短肽的发现。我们开发了第一种用于合成、筛选和测序富含半胱氨酸的OBOC肽文库的方法。首先，我们合成了一个高度偏向的富含半胱氨酸的OBOC文库，在每个位置掺入50%的半胱氨酸（Ac-X8-KM- Tentagel）。然后，我们开发了在单珠水平上对该文库进行半胱氨酸烷基化、溴化氰裂解和直接串联质谱测序的条件。该文库的测序效率与传统的无半胱氨酸文库相当。为了验证对富含半胱氨酸的OBOC文库的筛选，我们使一个文库与双砷荧光团FlAsH反应，并鉴定出带有已知双砷结合基序（CCXXCC）的珠子。这些结果使得OBOC文库能够用于高通量发现富含半胱氨酸的肽，用于蛋白质标记、金属污染物的环境修复或富含半胱氨酸的药物。
