Department of Chemistry, Indiana University, 800 East Kirkwood Avenue, Bloomington, Indiana 47405-7102, USA.
Anal Chem. 2011 Dec 1;83(23):9092-9. doi: 10.1021/ac202154r. Epub 2011 Oct 31.
Selective chemical modification of protein side chains coupled with mass spectrometry is often most informative when used to compare residue-specific reactivities in a number of functional states or macromolecular complexes. Herein, we develop ratiometric pulse-chase amidination mass spectrometry (rPAm-MS) as a site-specific probe of lysine reactivities at equilibrium using the Cu(I)-sensing repressor CsoR from Bacillus subtilis as a model system. CsoR in various allosteric states was reacted with S-methyl thioacetimidate (SMTA) for pulse time, t, and chased with excess of S-methyl thiopropionimidate (SMTP) (Δ = 14 amu), quenched and digested with chymotrypsin or Glu-C protease, and peptides were quantified by high-resolution matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry and/or liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). We show that the reactivities of individual lysines from peptides containing up to three Lys residues are readily quantified using this method. New insights into operator DNA binding and the Cu(I)-mediated structural transition in the tetrameric copper sensor CsoR are also obtained.
当用于比较许多功能状态或大分子复合物中残基特异性反应性时，与质谱联用的蛋白质侧链的选择性化学修饰通常是最具信息量的。在此，我们开发了比率脉冲-追踪氨甲酰化质谱（rPAm-MS），以作为使用来自枯草芽孢杆菌的 Cu(I)感应阻遏物 CsoR 的模型系统，在平衡时检测赖氨酸反应性的一种特异性探针。将 CsoR 处于各种变构状态下与 S-甲基硫代乙内酰脲（SMTA）反应进行脉冲时间 t，然后用过量的 S-甲基丙二酰亚胺（SMTP）（Δ = 14 amu）进行追踪，用胰凝乳蛋白酶或 Glu-C 蛋白酶猝灭和消化，并通过高分辨率基质辅助激光解吸电离飞行时间（MALDI-TOF）质谱和/或液相色谱电喷雾串联质谱（LC-ESI-MS/MS）定量肽。我们表明，使用该方法可以轻松定量包含多达三个 Lys 残基的肽中各个 Lys 的反应性。还获得了有关操作员 DNA 结合和四聚体铜传感器 CsoR 中 Cu(I)介导的结构转变的新见解。
