Radicalomics profile of RAW264.7 cells exposed to lipopolysaccharide (LPS) and/or 5,5-dimethyl-1-pyrroline N-oxide: a proteomics iTRAQ raw data set
Description
This study had as the main goal to identify proteins that become free radicals in RAW264.7 cells primed with LPS. This priming results in the production of reactive biochemical species that can cause radical formation in cell macromolecules, including in proteins. Like mechanisms of protein radicalization, Fenton chemistry and other electron-oxidation mechanisms may be involved. To trap protein radicals, we used the nitrone spin trap, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), which traps, in situ and in real time, those protein radicals formed inside the primed cells. The trapping of protein radicals by DMPO forms protein-DMPO nitrone adducts that can be detected by immuno-spin trapping (https://doi.org/10.1016/j.bbagen.2013.04.039). Immuno-spin trapping includes a number of techniques that can detect protein nitrone adducts, including western blot with an antibody anti-DMPO (commercially available). In this study, 14 bands of protein-DMPO nitrone adducts were detected by western blot with the antibody anti-DMPO. These bands were selected considering that nitrone adducts are formed in a concentration-dependent manner with the concentration of DMPO added to RAW264.7 cells primed with LPS (1ng/ml). These 14 bands were located in a 1-D SDS-PAGE stained with coomassie blue. Each of these bands was cut from these gels and proteins were digested and analyzed by MS (See full description in Step to reproduce). Results: Fourteen bands were cut from the gel Exp#85‐56. All of the bands were cut from the same lane (the fourth lane from the right). These bands were washed, reduced/alkylated, and digested with trypsin. The digest was analyzed by capillary column LC‐tandem MS and the CID spectra searched against the NCBI RefSeq database. The results are summarized in Table 2, with the specific peptides that were sequenced shown in the attached figures. This study resulted in the localization of several radicalized proteins in RAW264.7 cells primed with LPS (10.1016/j.freeradbiomed.2012.04.023), among them GAPDH (https://pmc.ncbi.nlm.nih.gov/articles/PMC11500056/), one of the most sensitive protein to redox modification in living cells. SOME CONSIDERATIONS TO TAKE INTO ACCOUNT WHEN INTERPRETING THESE DATA SET Each band, depending on the intensity of Coomassie blue staining, can have about 300 different proteins. Although 1D SDS-PAGE gel was run under reducing conditions, some polypeptide can be aggregated by hydrophobic interactions, or other non S-S-mediated protein cross-links. As a consequence of the above consideration, it may be that some protein-DMPO positive bands contain a minor protein in the aggregate that is forming a radical protein. Thus is possible that the protein that forms a radical can not be included in the list because is a minor abundance protein in the analyzed band. This would require further study of individual proteins in biochemical systems of oxidation in the presence of DMPO.
Files
Steps to reproduce
RAW264.7 cells were incubated in the presence or absence of 1 ng/ml LPS and different concentrations of DMPO (0, 10, 25 y 50 mM) in DMEM high glucose with 10% radiation-inactivated fetal calf serum for 18h. After incubation, cells were lyzed using RIPA buffer with protease inhibitors and Triton X100 as detergent. After protein adjustment (BCA Protein determination assay), proteins were resolved using -4-10% SDS-PAGE under reducing conditions. Proteins were blotted from the gel onto a nitrocelullose membrane. Protein-DMPO nitrone adducts (positive bands in the western blot) were determined by immuno-spin trapping as previously described (10.1002/0471140856.tx1707s24). The intensity of 14 bands observed in Western blot increased with the concentration of DMPO, suggesting that the protein-DMPO nitrone adduct requires generation of a radical site in the protein to be trapped by DMPO. The data set shows an image of the gel image where the 14 bands are labeled. These bands in the gel were cut and digested as indicated in the description. Brief description of samples and goal of the experiment The samples were a series of bands from a Coomassie blue‐stained 1D gel. Table 1 contains a summary of the samples analyzed in this set. A scan of the gel showing the specific bands of interest is also attached. Methods: For the protein digestion, the bands were cut from the gel as closely as possible with a scalpel and washed/destained in 50% ethanol, 5% acetic acid. The gel pieces were then reduced with DTT and alkylated with iodoacetamide before digestion with trypsin overnight. The peptides that were formed were extracted from the polyacrylamide and the extract evaporated to <30 µL for LC‐MS analysis. The LC‐MS system was a Thermo Scientific LTQ‐xl ion trap mass spectrometer system. The HPLC column was a self‐packed 8 cm x 75 µm id Phenomenex Jupiter Proteo reversed phase capillary chromatography column. Ten µL volumes of the extract were injected and the peptides eluted from the column by an acetonitrile/0.1% formic acid gradient at a flow rate of 200 nL/min. The digest was analyzed using the data dependent multitask capability of the instrument acquiring full scan mass spectra to determine peptide molecular weights and product ion spectra to determine amino acid sequence in successive instrument scans. This mode of analysis produces approximately 3000 collisionally‐induced dissociation (CID) spectra of ions ranging in abundance over several orders of magnitude. The data were analyzed by using all CID spectra collected in the experiment to search the NCBI databases with the search programs Mascot and Sequest. Generally, we search the RefSeq database for the species from which the samples are derived although some searches may use the entire database. Each identification is verified by manual inspection of several matching spectra.
Institutions
- CONICET San Luis
- Universidad Nacional de San Luis
Categories
Funders
- Agencia Nacional de Promoción de la Investigación, el Desarrollo Tecnológico y la InnovaciónArgentinaGrant ID: PICT-2021-I-A-00147