A Filtered Database Search Algorithm for Endogenous Serum Protein Carbonyl Modifications in a Mouse Model of Inflammation

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. Molecular & Cellular

Protein carbonylation as a major hallmark of oxidative damage: Update of analytical strategies

“…This significantly reduced the complexity of the dataset for subsequent database searches and hence increased the identification rates and reduced the false positive hits. The obtained dataset can be further analyzed for carbonylated peptides using a de novo ‐like sequencing database (Biotin Peptide Identification Program) that contains only protein sequences of interest (e.g., plasma proteome) (Slade et al, ).…”

“…Non‐specific binding of proteins/peptides to avidin columns via hydrophobic and/or electrostatic interactions and the enrichment of endogenously biotinylated proteins additionally complicates the analysis. Unmodified peptides have been identified in ARP‐labeled BSA digests, even after extensive washing (20 column volumes) (Slade et al, ). Such background can be filtered out by stable isotope labeling of affinity‐purified proteins, for example by multiplexed iTRAQ stable isotope labeling.…”

Protein modifications by electrophilic lipoxidation products: Adduct formation, chemical strategies and tandem mass spectrometry for their detection and identification

“…Slade et al developed a filtered database search strategy to aid in the identification of ARP‐tagged oxylipid‐modified peptides (Slade et al, ). An in silico filter program uses the ARP‐specific fragment ions to filter the tandem mass spectral data and create a set of new datafiles that only contain the oxylipid modified peptides.…”

“…Searching this curated dataset by MASCOT, a widely used software search engine that uses tandem mass spectrometry data to identify proteins from primary sequence databases, limits the number of false positive hits and simplifies the manual inspection of the MS/MS data. As an alternative to MASCOT, Slade et al also introduced a specialized database search engine for the identification of biotin‐containing peptides without the prerequisite of defining the masses of possible oxylipid modifications (Slade et al, ). This search and identification strategy identified serum proteins with diverse oxylipid modifications in a murine model of inflammation, for example, DODE adducts were assigned to specific residues on mouse serum albumin and HNE adducts were identified on multiple murinoglobulin sites (Slade et al, ).…”

AminoxyTMT: A novel Multi-Functional Reagent for Characterization of Protein Carbonylation

“…The reagent, aminoxyTMT, is a derivative of the popular, MS/MS-compatible Tandem Mass Tag (TMT) isobaric labeling reagents from Thermo Fisher Scientific that contains a carbonyl-reactive aminooxy group (26, 27). Focusing on the common carbonyl modification by HNE on protein side-chains, we demonstrate the effectiveness of aminoxyTMT for both immunoblotting to visualize protein carbonylation using the available anti-TMT antibody and enriching aminoxyTMT-labeled peptides from complex mixtures.…”