Abstract
During inflammation, the resulting oxidative stress can damage surrounding host tissue, forming protein-carbonyls. The SJL mouse is an experimental animal model used to assess in vivo toxicological responses to reactive oxygen and nitrogen species from inflammation. The goals of this study were to identify the major serum proteins modified with a carbonyl functionality and to identify the types of carbonyl adducts. To select for carbonyl-modified proteins, serum proteins were reacted with an aldehyde reactive probe that biotinylated the carbonyl modification. Modified proteins were enriched by avidin affinity and identified by two-dimensional liquid chromatography tandem MS. To identify the carbonyl modification, tryptic peptides from serum proteins were subjected to avidin affinity and the enriched modified peptides were analyzed by liquid chromatography tandem MS. It was noted that the aldehyde reactive probe tag created tag-specific fragment ions and neutral losses, and these extra features in the mass spectra inhibited identification of the modified peptides by database searching. To enhance the identification of carbonyl-modified peptides, a program was written that used the tag-specific fragment ions as a fingerprint (in silico filter program) and filtered the mass spectrometry data to highlight only modified peptides. A de novo-like database search algorithm was written (biotin peptide identification program) to identify the carbonyl-modified peptides. Although written specifically for our experiments, this software can be adapted to other modification and enrichment systems. Using these routines, a number of lipid peroxidation-derived protein carbonyls and direct side-chain oxidation proteins carbonyls were identified in SJL mouse serum.
Highlights
From the ‡Department of Biological Engineering, and §Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139
Increased protein carbonyl modification has been reported in studies of the colon mucosal lining from patients diagnosed with inflammatory bowel disease [20, 21]
We recently identified proteins modified by the carbonyl 9,12-dioxo-10(E)dodecenoic acid (DODE) in cells treated with the hydroperoxide of linoleic acid (13-HPODE) [24]
Summary
DNA modification because of inflammation has been the focus of many animal and human studies, it is proteins that are considered most likely to be ubiquitously affected by disease, response, and recovery [13], and the biological consequences include more rapid protein turnover as well as novel signaling (14 –16). Increased protein carbonyl modification has been reported in studies of the colon mucosal lining from patients diagnosed with inflammatory bowel disease [20, 21]. Taken together, these observations suggest that an increase. A number of DODE modified proteins were identified, we were unable to definitively identify the carbonyl modified peptides by mass spectrometry due both to low abundance and to the interference of ARP-tag-specific fragment ions on database searching
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