Leavell Michael D, Novak Petr, Behrens Christopher R, Schoeniger Joseph S, Kruppa Gary H
Sandia National Laboratories, Livermore, California 94551-0969, USA.
J Am Soc Mass Spectrom. 2004 Nov;15(11):1604-11. doi: 10.1016/j.jasms.2004.07.018.
Chemical cross-linking of proteins combined with mass spectral analysis is a powerful technique that can be utilized to yield protein structural information, such as the spatial arrangement of multi-protein complexes or the folding of monomeric proteins. The succinimidyl ester cross-linking reagents are commonly used to cross-link primary amine-containing amino acids (N-terminus and lysine). However, in this study they were used to react with tyrosines as well, which allowed for the formation of cross-links between two primary amines, one primary amine and one tyrosine, or two tyrosines. This result is extremely important to the chemical cross-linking community for two reasons: (1) all possible cross-linked residues must be considered when analyzing data from these experiments to generate correct distance constraints and structural information, and (2) utilizing the versatility of these cross-linking reagents allows more information content to be generated from a single cross-linking reagent, which may increase the number of cross-links obtained in the experiment. Herein, we study the reactivity of the succinimidyl ester labeling and cross-linking reagents with angiotensin I and oxidized insulin beta-chain. Using the succinimidyl acetate labeling reagent, the reactivity of the N-terminus was found to be greater than either lysine or tyrosine. However, a selectivity of the cross-linking reagent was observed for either tyrosine or lysine depending on the pH of the reaction solution. In acidic pH, it was observed that tyrosine was more reactive, while in alkaline pH lysine was more reactive. Exploiting this selectivity predominantly N-terminus-tyrosine or tyrosine-tyrosine cross-links were favored at acidic pH, while N-terminus-tyrosine or tyrosine-lysine cross-links were favored at alkaline pH.
蛋白质化学交联结合质谱分析是一种强大的技术，可用于获取蛋白质结构信息，例如多蛋白复合物的空间排列或单体蛋白的折叠情况。琥珀酰亚胺酯交联试剂通常用于交联含伯胺的氨基酸（N端和赖氨酸）。然而，在本研究中，它们也用于与酪氨酸反应，从而实现两个伯胺之间、一个伯胺与一个酪氨酸之间或两个酪氨酸之间的交联。这一结果对化学交联领域极为重要，原因有二：（1）在分析这些实验数据以生成正确的距离限制和结构信息时，必须考虑所有可能的交联残基；（2）利用这些交联试剂的多功能性可从单一交联试剂生成更多信息内容，这可能会增加实验中获得的交联数量。在此，我们研究了琥珀酰亚胺酯标记和交联试剂与血管紧张素I和氧化胰岛素β链的反应活性。使用乙酸琥珀酰亚胺酯标记试剂时，发现N端的反应活性高于赖氨酸或酪氨酸。然而，根据反应溶液的pH值，观察到交联试剂对酪氨酸或赖氨酸具有选择性。在酸性pH条件下，观察到酪氨酸反应性更高，而在碱性pH条件下赖氨酸反应性更高。利用这种选择性，在酸性pH条件下主要形成N端-酪氨酸或酪氨酸-酪氨酸交联，而在碱性pH条件下则主要形成N端-酪氨酸或酪氨酸-赖氨酸交联。
