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

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.