A top-down LC-FTICR MS-based strategy for characterizing oxidized calmodulin in activated macrophages |
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Authors: | Natacha Lourette Heather Smallwood Si Wu Errol W. Robinson Thomas C. Squier Richard D. Smith Ljiljana Paša-Tolić |
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Affiliation: | 1. Fundamental and Computational Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA 2. DSM Research Campus Geleen, Chemelot Gate 2/Routepunt 515, Urmonderbaan 22, 6167, RD Geleen, The Netherlands 3. Department of Immunology, St. Jude Children’s Research Hospital, 262 Danny Thomas Place/MS 351, 38105-3678, Memphis, TN, USA 4. Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, 902 Battelle Blvd., P.O. Box 999 (MSIN: K8-98), 99352, Richland, WA, USA
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Abstract: | 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 ~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. |
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