[en] A liquid chromatography-mass spectrometry (LC-MS)-based approach for characterizing the degree of nitration and oxidation of intact calmodulin (CaM) has been used to resolve approximately 250 CaM oxiforms using only 500 ng of protein. The analysis was based on high-resolution data of the intact CaM isoforms obtained by Fourier-transform ion cyclotron resonance mass spectrometry (FTICR MS) coupled with an on-line reversed-phase LC separation. Tentative identifications of post-translational modifications (PTMs), such as oxidation or nitration, have been assigned by matching observed protein mass to a database containing all theoretically predicted oxidation products of CaM and verified through a combination of tryptic peptide information (generated from bottom-up analyses) and on-line collisionally induced dissociation (CID) tandem mass spectrometry (MS/MS) at the intact protein level. The reduction in abundance and diversity of oxidatively modified CaM (i.e., nitrated tyrosines and oxidized methionines) induced by macrophage activation has been explored and semiquantified for different oxidation degrees (i.e., no oxidation, moderate, and high oxidation). This work demonstrates the power of the top-down approach to identify and quantify hundreds of combinations of PTMs for single protein target such as CaM and implicate competing repair and peptidase activities to modulate cellular metabolism in response to oxidative stress.
Disciplines :
Chemistry
Author, co-author :
Lourette, Natacha ✱; Fundamental and Computational Sciences Division, Pacific Northwest National Laboratory, Richland, Washington 99352, USA
Smallwood, Heather ✱; Fundamental and Computational Sciences Division, Pacific Northwest National Laboratory, Washington, United States
Wu, Si; Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Washington, United States
Robinson, Errol W; Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Washington, United States
Squier, Thomas C; Fundamental and Computational Sciences Division, Pacific Northwest National Laboratory, Washington, United States
Smith, Richard D; Fundamental and Computational Sciences Division, Pacific Northwest National Laboratory, Washington, United States
Pasa-Tolić, Ljiljana; Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, 902 Battelle Blvd., 99352, Richland, WA, USA
✱ These authors have contributed equally to this work.
Language :
English
Title :
A top-down LC-FTICR MS-based strategy for characterizing oxidized calmodulin in activated macrophages.
Publication date :
June 2010
Journal title :
Journal of the American Society for Mass Spectrometry
Portions of this work were supported by the National Center for Research Resources ( RR 018522 ) and the U.S. Department of Energy (DOE) Office of Biological and Environmental Research. Work was performed in the Environmental Molecular Science Laboratory, a DOE national scientific user facility located on the campus of Pacific Northwest National Laboratory (PNNL) in Richland, Washington. PNNL is a multi-program national laboratory operated by Battelle for the DOE under contract DE-AC05-76RLO 1830.
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