RESUMO
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-tandem mass spectrometry (LC-MS/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 "steer" 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.
Assuntos
Cromatografia Líquida de Alta Pressão , Metano/análogos & derivados , Peptídeos/análise , Espectrometria de Massas em Tandem , Sequência de Aminoácidos , Metano/química , Fotólise , Pegadas de Proteínas , Eletricidade EstáticaRESUMO
Mass spectrometry is an important technology for mapping composition and flux in whole proteomes. Over the last 5 years in particular, impressive gains in the depth of proteome coverage have been realized, particularly for model organisms. This review will provide an update on advancements in the key analytical techniques, methods and informatics directed towards whole proteome analysis by mass spectrometry. Practical issues involving sample requirements, analysis time and depth of coverage will be addressed, to gauge how useful data-driven approaches are for solving biological problems. Targeted mass spectrometric methods, based on selected reaction monitoring, are presented as a powerful alternative to data-driven methods. They offer robust, transferable protocols for hypothesis-directed monitoring of limited yet biologically significant tracts of any proteome.