Biotinylated analogue of the spin-trap 5,5-dimethyl-1-pyrroline-N-oxide for the detection of low-abundance protein radicals by mass spectrometry

Abstract

Protein radicals are implicated in oxidative stress and are associated with a wide range of diseases and disorders. In the present work, we describe the specific application of a newly synthesized nitrone spin trap, Bio-SS-DMPO, for the detection of these highly reactive species by mass spectrometry (MS). Bio-SS-DMPO is a biotinylated analogue of the spin-trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) that allows for specific capture of the protein(s)/peptide(s) labeled by the spin-trap on a (strept)avidin-bound solid matrix. The disulfide bond in the linker arm joining biotin to DMPO can be cleaved to release captured spin-adduct peptide from the solid matrix. This (strept)avidin-based affinity purification reduces the complexity of the samples prior to MS analyses, thereby facilitating the location of the sites of spin trap addition. In addition, the biotin moiety on the spin-trap can efficiently be probed with (strept)avidin-conjugated reporter. This offers an effective means to visualize the presence of DMPO-adducted proteins in intact cells.

Figure 1

Chemical structure of Bio-SS-DMPO, a biotinylated analog of the spin-trap DMPO containing a chemically cleavable disulfide bond in the linker arm joining biotin to DMPO.

Figure 2

Deconvoluted electrospray mass spectrum obtained from the reaction of horse metmyoglobin with hydrogen peroxide in the presence of Bio-SS-DMPO.

Figure 3

BPI chromatograms acquired during the LC-ESI/MS analysis of the horse heart myoglobin/H₂O₂/Bio-SS-DMPO tryptic digest (A) prior to purification with streptavidin beads, and (B) after capture and release of DMPO-adducted peptides. Two abundant ions detected in Peak 1 correspond in mass to tryptic peptide T16 modified by the spin trap. The low abundance peaks (<5% relative abundance) at 65–75 min correspond to minimal nonspecific binding of myoglobin tryptic peptides to the streptavidin beads.

Figure 4

MS/MS of the peptide released from the streptavidin beads allow identification of the site of DMPO adduction. The spectrum was acquired from the ion of m/z 543.5 (+4) which corresponds in mass to tryptic peptide T16 plus a S-carboxyamidomethyl-modified DMPO tag.

Figure 5

Confocal fluorescence imaging studies of the distribution of Bio-SS-DMPO in Raw-264.7 cells. (A) Cells were treated with 25 mM Bio-SS-DMPO for 6 hrs at 37°C; (B) same as A, but in the absence of Bio-SS-DMPO. Alexa 488 (green) was used to visualize the accumulation of Bio-SS-DMPO in the cells. 4’,6-diamino-2-phenylindole (DAPI, in blue) was used to visualize nuclei. Differential interference contrast microscopy (DIC, in gray) was used to enhance contrast between phases by polarized light.