Department of Chemistry and Chemical Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York 12180, USA.
J Am Soc Mass Spectrom. 2009 Nov;20(11):1974-82. doi: 10.1016/j.jasms.2009.08.002. Epub 2009 Aug 12.
The insulin-linked polymorphic region (ILPR) of the human insulin gene promoter region forms G-quadruplex structures in vitro. Previous studies show that insulin and insulin-like growth factor-2 (IGF-2) exhibit high affinity binding in vitro to 2-repeat sequences of ILPR variants a and h, but negligible binding to variant i. Two-repeat sequences of variants a and h form intramolecular G-quadruplex structures that are not evidenced for variant i. Here we report on the use of protein digestion combined with affinity capture and MALDI-MS detection to pinpoint ILPR binding sites in insulin and IGF-2. Peptides captured by ILPR variants a and h were sequenced by MALDI-MS/MS, LC-MS and in silico digestion. On-bead digestion of insulin-ILPR variant a complexes supported the conclusions. The results indicate that the sequence VCG(N)RGF is generally present in the captured peptides and is likely involved in the affinity binding interactions of the proteins with the ILPR G-quadruplexes. The significance of arginine in the interactions was studied by comparing the affinities of synthesized peptides VCGERGF and VCGEAGF with ILPR variant a. Peptides from other regions of the proteins that are connected through disulfide linkages were also detected in some capture experiments. Identification of binding sites could facilitate design of DNA binding ligands for capture and detection of insulin and IGF-2. The interactions may have biological significance as well.
胰岛素基因启动子区域的胰岛素连接多态性区域（ILPR）在体外形成 G-四链体结构。先前的研究表明，胰岛素和胰岛素样生长因子-2（IGF-2）在体外对 ILPR 变体 a 和 h 的 2 重复序列表现出高亲和力结合，但对变体 i 几乎没有结合。变体 a 和 h 的 2 重复序列形成分子内 G-四链体结构，而变体 i 则没有证据表明存在这种结构。在这里，我们报告了使用蛋白质消化结合亲和捕获和 MALDI-MS 检测来确定胰岛素和 IGF-2 中 ILPR 的结合位点。通过 MALDI-MS/MS、LC-MS 和计算机消化对 ILPR 变体 a 和 h 捕获的肽进行测序。在珠上消化胰岛素-ILPR 变体 a 复合物支持了这些结论。结果表明，序列 VCG（N）RGF 通常存在于捕获的肽中，并且可能参与了蛋白质与 ILPR G-四链体的亲和力结合相互作用。通过比较合成肽 VCGERGF 和 VCGEAGF 与 ILPR 变体 a 的亲和力，研究了精氨酸在相互作用中的意义。通过二硫键连接的蛋白质的其他区域的肽也在一些捕获实验中被检测到。结合位点的鉴定可以促进用于捕获和检测胰岛素和 IGF-2 的 DNA 结合配体的设计。这些相互作用可能具有生物学意义。
