c fragments T₁₂ and T₁₃ were further investigated by MS² and MS³ experiments.

The peaks at retention times of 45.87 min and 49.34 min have m/z values corresponding to an oxidized form of T₁₂. This peptide is of particular interest because it contains two possible oxidation sites, Met₁₀₉ and Met₁₂₄.


Figure 1. Base peak mass spectrum of tryptic fragments of oxidized CaM separated on a C8 Hypurity column and detected with Data Dependent ion trap MS/MS.


Table 1: Theoretical monoisotopic masses of peptide fragments compared to measured m/z values recovered from a tryptic digest of oxidized CaM by full-scan Data Dependent LC-MS/MS.

The full-scan MS² spectrum of the doubly charged ion at m/z 1209.5 for the peak at RT = 45.87 min shows a series of corresponding y and b ions for a MetSO-containing T₁₂ peptide (Figure 2A). The appearance of MetSO-specific neutral loss sequence ions (SOCH⁴) for the y ion series is consistent with the sulfoxide located on the Cterminal portion of the T₁₂ peptide (see insert, Figure 2A). Analysis of the most abundant peak (m/z 1177.4) in the MS² spectrum by MS³ confirms oxidation of Met-124 by detection of MetSOspecific neutral loss sequence ions of C-terminal origin (Figure 2b).


Figure 2A. Full-scan MS² spectrum of doubly-charged ion at m/z 1209.5 for peak at RT 45.87 min.
Insert: Amino acid sequence of T₁₂ fragment indicating major C- and Nterminal fragment ions detected by full-scan MS². '"/>

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