Abstract
Proteins present in cellular environments with high levels of reactive oxygen and nitrogen species and/or low levels of antioxidants are highly susceptible to oxidative post-translational modification (PTM). Irreversible oxidative PTMs can generate a complex distribution of modified protein molecules, recently termed as proteoforms. Using ubiquitin as a model system, we mapped oxidative modification sites using trypsin, Lys-C, and Glu-C peptides. Several M+16 Da proteoforms were detected as well as proteoforms that include other previously unidentified oxidative modifications. This work highlights the use of multiple protease digestions to give insights to the complexity of oxidative modifications possible in bottom-up analyses.
Highlights
Reactive oxygen and nitrogen species in cellular environments can result in macromolecular damage [1]
This is a factor of ten increase in M+16 Da ions in comparison to solutions containing only untreated ubiquitin
The masses of the deconvoluted native and M+16 Da peaks are 8564.601 and 8580.592 Da. These values are
Summary
Reactive oxygen and nitrogen species in cellular environments can result in macromolecular damage [1]. Protein tertiary structure can be modified as a result of oxidative attack and lead to loss or gain of protein function [2]. Site-specific modifications can include the incorporation of oxygen atoms to amino acid side chains (e.g., methionine, phenylalanine, tyrosine, leucine, histidine), conversion to aldehydes, ketones, or carboxylic acids, addition of nitrogen containing groups (e.g., R-NO, R-NO2), formation of advanced glycation end products (e.g. imidazolinones, pyrralines), and introduction of lipid peroxidation products (e.g., 4-hydroxy-transnonenal, malondialdehyde) [3,4,5]. Proteoforms can include molecules that arise due to the same post-translational modification (PTM) occurring at different amino acid residue positions in the protein. A protein that incorporates a single oxygen atom during a free radical attack from hydrogen peroxide (H2O2) or superoxide anion, may exist in multiple locations. Liquid chromatography (LC) or electrophoresis separations may be able to resolve the two proteoforms (i.e., A and B), multiple dissociation methods such as collision activation dissociation (CAD) [11], infrared multiphoton dissociation [12], PLOS ONE | DOI:10.1371/journal.pone.0116606 March 16, 2015
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