High-Resolution Mapping of Carbene-Based Protein Footprints

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• 作者：Chanelle C. Jumper ; Ryan Bomgarden ; John Rogers ; Chris Etienne ; David C. Schriemer
• 刊名：Analytical Chemistry
• 出版年：2012
• 出版时间：May 15, 2012
• 年：2012
• 卷：84
• 期：10
• 页码：4411-4418
• 全文大小：359K
• 年卷期：v.84,no.10(May 15, 2012)
• ISSN：1520-6882

文摘

Carbene chemistry has been used recently in structural mass spectrometry as a labeling method for mapping protein surfaces. The current study presents a method for quantitating label distribution at the amino acid level and explores the nature and basis for an earlier observation of labeling bias. With the use of a method based on liquid chromatography鈥搕andem mass spectrometry (LC鈥揗S/MS) applied to digests of holo-calmodulin, we developed a quantitation strategy to map site-specific incorporation of carbene, generated from photolysis of ionic label precursors 2-amino-4,4-azipentanoic acid and 4,4-azipentanoic acid. The approach provides reliable incorporation data for fragments generated by electron-transfer dissociation, whereas high-energy collisional dissociation leads to energy and sequence-dependent loss of the label as a neutral. However, both can produce data suitable for mapping residues in the interaction of holo-calmodulin with M13 peptide ligand. Site-specific labeling was monitored as a function of reagent, ionic strength, and temperature, demonstrating that electrostatic interactions at the protein surface can 鈥渟teer鈥?the distribution of label precursors to sites of surface charge and favor label insertion into residues in the vicinity of the surface charge. A further preference for insertion into carboxylates was observed, based on chemical reactivity. We suggest that decoupling surface partitioning from the chemistry of insertion offers a flexible, tunable labeling strategy for structural mass spectrometry that can be applied to a broad range of protein surface compositions and promotes the design of reagents to simplify the workflow.