Supplemental Figure 2. . out File Generated by Sequest for MS2 Spectrum in Figure 4B

Supplemental Figure 1. HNE-modified peptides from non-labeled and 18O-labeled rat skeletal muscle protein homogenate

A tryptic digest of HNE-treated rat skeletal muscle protein homogenate was enriched using the solid-phase hydrazide reagent (SPH), split in half and either non-labeled or labeled with 18O, respectively. The two fractions were submitted to Sequest database searching using accurate mass parameters. The identified HNE-modified peptides within 8ppm mass accuracy were then compared. The 8ppm delta mass constraint provided a 0% FPR for the 18O-labeled sample and a 0.8% FPR for the non-labeled control.

Supplemental Figure 2. .Out file generated by Sequest for MS2 spectrum in figure 4B

Supplemental Figure 3. MS and MS2 spectra confidently matched to a non-HNE modified peptide

The accuracy of the match is supported by both the delta mass of the precursor-ion (590.28m/z) in the MS spectrum (A), and by the alignment of the major product ions in the MS2 spectrum (B) with the expected series of b and y-ions for the peptide sequence. As expected for a non-modified peptide, both the precursor ion and all product ions generate typical isotope patterns free of 18O isotopologues.

Supplemental Figure 4. .Out file generated by Sequest for MS2 spectrum in figure 5B

Supplemental Figure 5. Representative MS spectrum showing inefficiency of the MS2 and database search platform for correctly identifying 18O-labeled peptides.

The isotope envelopes for each of the 18O-labeled ions observed over the defined m/z range are inlayed above the spectrum. The presence of an additional isotopologue observed in the envelope for three of the precursor-ions pictured (478.77, 486.78, and 508.78m/z) suggests that an additional 18O molecule was incorporated into the peptides. Further scrutiny of the MS2 spectra deriving from these three precursor-ions indicated that the additional isotopologue most likely resulted from incomplete reduction of the carbonyl or the presence of multiple modifications (data not shown). *, an MS2 spectrum was not acquired. ^, the MS2 spectrum acquired was not matched to a carbonylated peptide. $, the MS2 spectrum acquired matched to a carbonylated peptide, +, background peptide not labeled with 18O or matched to a carbonylated peptide by the database search.

Supplemental Figure 6. .Out file generated by Sequest for MS2 spectrum in supplemental figure 3B