Guo Xin, Bandyopadhyay Pradipta, Schilling Birgit, Young Malin M, Fujii Naoaki, Aynechi Tiba, Guy R Kiplin, Kuntz Irwin D, Gibson Bradford W
Department of Pharmaceutics and Medicinal Chemistry, University of the Pacific, Stockton, CA 95211, USA.
Anal Chem. 2008 Feb 15;80(4):951-60. doi: 10.1021/ac701636w. Epub 2008 Jan 18.
Intramolecular cross-linking coupled with mass spectrometric identification of cross-linked amino acids is a rapid method for elucidating low-resolution protein tertiary structures or fold families. However, previous cross-linking studies on model proteins, such as cytochrome c and ribonuclease A, identified a limited number of peptide cross-links that are biased toward only a few of the potentially reactive lysine residues. Here, we report an approach to improve the diversity of intramolecular protein cross-linking starting with a systematic quantitation of the reactivity of lysine residues of a model protein, bovine cytochrome c. Relative lysine reactivities among the 18 lysine residues of cytochrome c were determined by the ratio of d0 and acetyl-d3 groups at each lysine after partial acetylation with sulfosuccinimidyl acetate followed by denaturation and quantitative acetylation of remaining unmodified lysines with acetic-d6 anhydride. These lysine reactivities were then compared with theoretically derived pKa and relative solvent accessibility surface values. To ascertain if partial N-acetylation of the most reactive lysine residues prior to cross-linking can redirect and increase the observable Lys-Lys cross-links, partially acetylated bovine cytochrome c was cross-linked with the amine-specific, bis-functional reagent, bis(sulfosuccinimidyl)suberate. After proteolysis and mass spectrometry analysis, partial acetylation was shown to significantly increase the number of observable peptides containing Lys-Lys cross-links, shifting the pattern from the most reactive lysine residues to less reactive ones. More importantly, these additional cross-linked peptides contained novel Lys-Lys cross-link information not seen in the non-acetylated protein and provided additional distance constraints that were consistent with the crystal structure and facilitated the identification of the proper protein fold.
分子内交联结合交联氨基酸的质谱鉴定是阐明低分辨率蛋白质三级结构或折叠家族的快速方法。然而，先前对细胞色素c和核糖核酸酶A等模型蛋白的交联研究发现，有限数量的肽交联仅偏向少数几个潜在反应性赖氨酸残基。在这里，我们报告一种方法来提高分子内蛋白质交联的多样性，该方法始于对模型蛋白牛细胞色素c赖氨酸残基反应性的系统定量。通过用乙酰基琥珀酰亚胺磺酸酯进行部分乙酰化，然后使剩余未修饰的赖氨酸变性并用乙酸-d6酐进行定量乙酰化后，每个赖氨酸处d0和乙酰-d3基团的比率来确定细胞色素c的18个赖氨酸残基之间的相对赖氨酸反应性。然后将这些赖氨酸反应性与理论推导的pKa和相对溶剂可及性表面值进行比较。为了确定在交联之前最具反应性的赖氨酸残基的部分N-乙酰化是否可以重新定向并增加可观察到的Lys-Lys交联，将部分乙酰化的牛细胞色素c与胺特异性双功能试剂双（琥珀酰亚胺基）辛二酸酯交联。经过蛋白酶解和质谱分析，结果表明部分乙酰化可显著增加含有Lys-Lys交联的可观察到的肽的数量，使模式从最具反应性的赖氨酸残基转变为反应性较低的残基。更重要的是，这些额外的交联肽包含了未乙酰化蛋白中未见的新的Lys-Lys交联信息，并提供了与晶体结构一致的额外距离限制，有助于识别正确的蛋白质折叠。
