Table 1.

Criteria for 3-Nitrotyrosine (3NT) peptide identification by manual inspection of MS/MS spectra. Criteria were used to asses both QSTAR Elite (QTOF) and LTQ Velos (ion trap) spectra.

Abundant fragment ions need to be assigned Not more than 1 unassigned fragment ion over 50% of base peak intensity Similar MS/MS fragmentation pattern as unmodified peptide# At least 3 of the top 5 fragment ions in the unmodified and 3NT modified spectra correspond Chromatographic elution time of 3NT peptide is later than unmodified peptide# Presence of 3NT immonium ion with S/N >6 3-nitrotyrosine residues can form a characteristic immonium ion at 181.1 m/z. Flanking MS/MS fragment ions for 3NT site Presence of ≥ 1 fragment ion in the MS/MS spectrum that includes the modified tyrosine and has a resulting mass shift due to the 3NT modification as well as ≥ 1 fragment ion that does not include the modified Y and does not show a mass shift. Correct precursor ion charge state (QSTAR Elite only) Correct monoisotopic precursor mass chosen (QSTAR Elite only)$ Precursor mass accuracy < 25 ppm (QSTAR Elite only)

^#Corresponding unmodified peptides were identified on the QSTAR Elite for 70.4% of observed 3NT peptides and on the LTQ Velos for 78.7% of detected 3NT peptides.

^$Criteria applicable to QSTAR Elite data only since LTQ Velos data is searched using average mass of the precursor instead of the monoisotopic mass.