Mass spectrometric determination of ILPR G-quadruplex binding sites in insulin and IGF-2.
Mass spectrometric determination of ILPR G-quadruplex binding sites in insulin and IGF-2.
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DOI：
10.1016/j.jasms.2009.08.002
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发表时间：
2009-11
影响因子：
3.2
通讯作者：
McGown LB
中科院分区：
文献类型：
--
作者：
Xiao J;McGown LB
人胰岛素基因启动子区的胰岛素连锁多态性区（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）在体外表现出与ILPR变体a和h的2-重复序列的高亲和力结合，但与变体i的结合可忽略不计。变体a和h的两个重复序列形成分子内G-四链体结构，这在变体i中没有得到证实。在这里，我们报告使用蛋白质消化结合亲和捕获和MALDI-MS检测，以查明胰岛素和IGF-2的ILPR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。胰岛素-ILPR变体a复合物的珠上消化支持该结论。结果表明，序列VCG（N）RGF通常存在于捕获的肽中，并且可能参与蛋白质与ILPR G-四链体的亲和结合相互作用。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。在一些捕获实验中还检测到来自通过二硫键连接的蛋白质的其他区域的肽。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。这种相互作用也可能具有生物学意义。
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.
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影响因子：
30.8
作者：
Vafiadis, P;Bennett, ST;Polychronakos, C
通讯作者：
Polychronakos, C
影响因子：
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作者：
Cole, Jacquelyn R.;Dick, Lawrence W., Jr.;McGown, Linda B.
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McGown, Linda B.
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通讯作者：
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