Sinz Andrea, Kalkhof Stefan, Ihling Christian
Biotechnological-Biomedical Center, Faculty of Chemistry and Mineralogy, University of Leipzig, Germany.
J Am Soc Mass Spectrom. 2005 Dec;16(12):1921-31. doi: 10.1016/j.jasms.2005.07.020. Epub 2005 Oct 24.
Chemical cross-linking of protein complexes has gained renewed interest in combination with mass spectrometric analysis of the reaction products as it allows a rapid mapping of protein interfaces, which is crucial for understanding protein/protein interactions. The identification of cross-linking products from the complex mixtures created after the cross-linking reaction, however, remains a daunting task. To facilitate the identification of cross-linking products, we explore the use of the commercially available biotinylated cross-linking reagent sulfo-SBED (sulfosuccinimidyl-2-[6-(biotinamido)-2-(p-azidobenzamido)-hexanoamido]ethyl-1,3'-dithiopropionate). This trifunctional cross-linker possesses one amine-reactive and one photo-reactive site and, additionally, allows an affinity-based enrichment of cross-linker containing species. As a model system, we chose the Ca(2+)-dependent complex between calmodulin and its target peptide M13, which represents a part of the C-terminal sequence of the skeletal muscle myosin light chain kinase. After the cross-linking reaction, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and one-dimensional gel electrophoresis were employed to check for the extent of cross-linking product formation. The cross-linking reaction mixtures were subjected to tryptic in-solution digestion. Biotinylated peptides, e.g., peptides that had been modified by the cross-linker as well as cross-linked peptides, were enriched on monomeric avidin beads after several washing steps had been performed. Peptide mixtures were analyzed by MALDI-TOFMS, nano-high-performance liquid chromatography (HPLC)/nano-electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICRMS), and tandem MS. We demonstrate that an enrichment of cross-linker containing species allows a more efficient identification of interacting amino acid sequences in protein complexes. This strategy is expected to be especially beneficial for investigating large protein assemblies.
蛋白质复合物的化学交联与反应产物的质谱分析相结合，重新引起了人们的兴趣，因为它可以快速绘制蛋白质界面图谱，这对于理解蛋白质/蛋白质相互作用至关重要。然而，从交联反应后产生的复杂混合物中鉴定交联产物仍然是一项艰巨的任务。为了便于鉴定交联产物，我们探索使用市售的生物素化交联试剂磺基-SBED（磺基琥珀酰亚胺基-2-[6-(生物素酰胺基)-2-(对叠氮苯甲酰胺基)-己酰胺基]乙基-1,3'-二硫代丙酸酯）。这种三功能交联剂具有一个胺反应性位点和一个光反应性位点，此外，还允许基于亲和力富集含交联剂的物种。作为一个模型系统，我们选择了钙调蛋白与其靶肽M13之间的Ca(2+)依赖性复合物，M13代表骨骼肌肌球蛋白轻链激酶C端序列的一部分。交联反应后，采用基质辅助激光解吸/电离飞行时间质谱（MALDI-TOFMS）和一维凝胶电泳来检查交联产物的形成程度。交联反应混合物进行胰蛋白酶溶液内消化。经过几个洗涤步骤后，生物素化肽，例如被交联剂修饰的肽以及交联肽，在单体抗生物素蛋白珠上富集。肽混合物通过MALDI-TOFMS、纳升级高效液相色谱（HPLC）/纳升电喷雾电离傅里叶变换离子回旋共振质谱（ESI-FTICRMS）和串联质谱进行分析。我们证明，富集含交联剂的物种可以更有效地鉴定蛋白质复合物中相互作用的氨基酸序列。预计该策略对于研究大型蛋白质组装体特别有益。
