Protein surface accessible residues play an important role in protein folding, protein-protein interactions and protein-ligand binding. However, a common problem associated with the use of selective chemical labeling methods for mapping protein solvent accessible residues is that when a complicated peptide mixture resulting from a large protein or protein complex is analyzed, the modified peptides may be difficult to identify and characterize amongst the largely unmodified peptide population (i.e., the ‘needle in a haystack’ problem). To address this challenge, we describe here the development of a strategy involving the synthesis and application of a novel ‘fixed charge’ sulfonium ion containing lysine-specific protein modification reagent, S,S′-dimethylthiobutanoylhydroxysuccinimide ester (DMBNHS), coupled with capillary HPLC-ESI-MS, automated CID-MS/MS, and data-dependant neutral loss mode MS³ in an ion trap mass spectrometer, to map the surface accessible lysine residues in a small model protein, cellular retinoic acid binding protein II (CRABP II). After reaction with different reagent:protein ratios and digestion with Glu-C, modified peptides are selectively identified and the number of modifications within each peptide are determined by CID-MS/MS, via the exclusive neutral loss(es) of dimethylsulfide, independently of the amino acid composition and precursor ion charge state (i.e., proton mobility) of the peptide. The observation of these characteristic neutral losses are then used to automatically ‘trigger’ the acquisition of an MS³ spectrum to allow the peptide sequence and the site(s) of modification to be characterized. Using this approach, the experimentally determined relative solvent accessibilities of the lysine residues were found to show good agreement with the known solution structure of CRABP II.

蛋白质表面可及残基在蛋白质折叠、蛋白质-蛋白质相互作用和蛋白质-配体结合中起着重要作用。然而，与使用选择性化学标记方法绘制蛋白质溶剂可及残基图谱相关的一个常见问题是，当分析由大蛋白质或蛋白质复合物产生的复杂肽混合物时，修饰的肽可能难以识别和表征。大部分未修饰的肽群（即“大海捞针”问题）。为了应对这一挑战，我们在此描述了一种策略的开发，该策略涉及一种新型“固定电荷”锍离子的合成和应用，该锍离子含有赖氨酸特异性蛋白质修饰试剂 S,S'-二甲基硫代丁酰基羟基琥珀酰亚胺酯 (DMBNHS)，并结合毛细管 HPLC -ESI-MS，³在离子阱质谱仪中，绘制小模型蛋白质细胞视黄酸结合蛋白 II (CRABP II) 中表面可接近的赖氨酸残基。在与不同的试剂：蛋白质比例反应并用 Glu-C 消化后，修饰肽被选择性识别，每个肽内的修饰数量由 CID-MS/MS 确定，通过二甲基硫醚的独家中性损失，独立于肽的氨基酸组成和前体离子电荷状态（即质子迁移率）。然后使用对这些特征中性损耗的观察来自动“触发”MS ³谱以允许表征肽序列和修饰位点。使用这种方法，发现赖氨酸残基的实验确定的相对溶剂可及性与已知的 CRABP II 溶液结构非常吻合。