Table 1.
Site | Peptide | Native (Area) | N15-labeled(Area) | Modified (%) |
---|---|---|---|---|
C46 | DPVQCSR | 14,214,288 | 15,332,719 | 7.3 |
C53 | DVVICPDASLEDAK | 69,277,937 | 229,809,312 | 70.0 |
C106 | GLIAAICAGPTALLAHEIGFGSK | 268,610,760 | 1,179,067,215 | 77.2A |
Quantitation of estimated glutathionylation of cysteines of DJ-1. Glutathionylation of cysteine residues 46, 53, and 106 were estimated using unmodified 15N DJ-1 as an internal standard. Quantification of modified tryptic peptides were estimated by subtraction of the peak areas of the non-modified peptides in the treated 14N-samples from those in the untreated 15N-samples (Supplemental Figure 3).
Example of calculation of extent of glutathionylation of Cys106: 1,179,067,215 (15N non-modified area) - 268,610,760 (14N non-modified area) = 910,456,455 (estimated area of the glutathionylated peptide). 910,456,455 (estimated are of the glutathionylated peptide) / 1,179,067,215 (15N non-modified area) × 100 = 77.2% (Estimated percentage of glutathionylated peptide as compared to total internal standard).