Mass spectrometric determination of ILPR G-quadruplex binding sites in insulin and IGF-2.
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.
Xiao
JunFeng
J
Department of Chemistry and Chemical Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York 12180, USA.
McGown
Linda B
LB
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D019921:Biotinylation; D053818:Chymases / Q000235:genetics / Q000378:metabolism; D054856:G-Quadruplexes; D006801:Humans; D007328:Insulin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007335:Insulin-Like Growth Factor II / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis / Q000138:chemical synthesis / Q000737:chemistry; D011110:Polymorphism, Genetic; D011401:Promoter Regions, Genetic; D011485:Protein Binding / Q000235:genetics; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D007328:Insulin; D009841:Oligonucleotides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D007335:Insulin-Like Growth Factor II; D053818:Chymases