High throughput protein fold identification by using experimental constraints derived from intramolecular cross-links and mass spectrometry

We have used intramolecular cross-linking, MS, and sequence threading to rapidly identify the fold of a model protein, bovine basic fibroblast growth factor (FGF)-2. Its tertiary structure was probed with a lysine-specific cross-linking agent, bis(sulfosuccinimidyl) suberate (BS(3)). Sites of cross-linking were determined by tryptic peptide mapping by using time-of-flight MS. Eighteen unique intramolecular lysine (Lys-Lys) cross-links were identified. The assignments for eight cross-linked peptides were confirmed by using post source decay MS. The interatomic distance constraints were all consistent with the tertiary structure of FGF-2. These relatively few constraints, in conjunction with threading, correctly identified FGF-2 as a member of the β-trefoil fold family. To further demonstrate utility, we used the top-scoring homolog, IL-1β, to build an FGF-2 homology model with a backbone error of 4.8 Å (rms deviation). This method is fast, is general, uses small amounts of material, and is amenable to automation.

本研究采用分子内交联（intramolecular cross-linking）、质谱（MS）与序列穿线法（sequence threading），快速鉴定了模式蛋白——牛碱性成纤维细胞生长因子（FGF）-2的折叠类型。我们使用赖氨酸特异性交联剂双(磺基琥珀酰亚胺基)辛二酰亚胺（BS3）对其三级结构进行探测，通过胰蛋白酶肽谱分析结合飞行时间质谱（time-of-flight MS）确定交联位点，共鉴定出18个独特的分子内赖氨酸（Lys-Lys）交联位点；通过源后衰变质谱（post source decay MS）验证了其中8个交联肽段的位点归属。所得原子间距离约束均与FGF-2的三级结构相符，凭借这些数量相对较少的约束条件结合序列穿线分析，我们准确将FGF-2归类为β-三叶折叠（β-trefoil fold）家族成员。为进一步验证该方法的实用性，我们以得分最高的同源蛋白白细胞介素-1β（IL-1β）为模板，构建了FGF-2的同源建模模型，其主链均方根偏差（rms deviation）为4.8埃。该方法快速通用、样品需求量少且易于实现自动化。