Golden M C, Resing K A, Collins B D, Willis M C, Koch T H
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215, USA.
Protein Sci. 1999 Dec;8(12):2806-12. doi: 10.1110/ps.8.12.2806.
A photocrosslink between basic fibroblast growth factor (bFGF155) and a high affinity ssDNA oligonucleotide was characterized by positive ion electrospray ionization mass spectrometry (ESIMS). The DNA was a 61-mer oligonucleotide photoaptamer bearing seven bromodeoxyuridines, identified by in vitro selection. Specific photocrosslinking of the protein to the oligonucleotide was achieved by 308 nm XeCl excimer laser excitation. The cross-linked protein nucleic acid complex was proteolyzed with trypsin. The resulting peptide crosslink was purified by PAGE, eluted, and digested by snake venom phosphodiesterase/alkaline phosphatase. Comparison of the oligonucleotide vs. the degraded peptide crosslink by high performance liquid chromatography coupled to an electrospray ionization triple quadrupole mass spectrometer showed a single ion unique to the crosslinked material. Sequencing by collision induced dissociation (MS/MS) on a triple quadrupole mass spectrometer revealed that this ion was the nonapeptide TGQYKLGSK (residues 130-138) crosslinked to a dinucleotide at Tyr133. The MS/MS spectrum indicated sequential fragmentation of the oligonucleotide to uracil covalently attached to the nonapeptide followed by fragmentation of the peptide bonds. Tyr133 is located within the heparin binding pocket, suggesting that the in vitro selection targeted this negative ion binding region of bFGF155.
通过正离子电喷雾电离质谱（ESIMS）对碱性成纤维细胞生长因子（bFGF155）与高亲和力单链DNA寡核苷酸之间的光交联进行了表征。该DNA是一种带有七个溴脱氧尿苷的61聚体寡核苷酸光适配体，通过体外筛选鉴定。通过308 nm XeCl准分子激光激发实现了蛋白质与寡核苷酸的特异性光交联。用胰蛋白酶对交联的蛋白质核酸复合物进行蛋白酶解。通过聚丙烯酰胺凝胶电泳（PAGE）纯化所得的肽交联物，洗脱后用蛇毒磷酸二酯酶/碱性磷酸酶消化。通过高效液相色谱与电喷雾电离三重四极杆质谱联用比较寡核苷酸与降解的肽交联物，结果显示交联材料有一个独特的单离子。在三重四极杆质谱仪上通过碰撞诱导解离（MS/MS）进行测序，结果表明该离子是与二核苷酸在Tyr133处交联的九肽TGQYKLGSK（残基130 - 138）。MS/MS谱图表明寡核苷酸依次断裂为与九肽共价连接的尿嘧啶，随后肽键断裂。Tyr133位于肝素结合口袋内，这表明体外筛选靶向了bFGF155的这个负离子结合区域。
