( rmo Hypersil-Keystone) at a flow rate ...)Identification of Low-Level Oxidation Products in Calmodulin Using NanoSpray ,,, Ion Trap Mass Spectrometry

rmo Hypersil-Keystone) at a flow rate of 700 nL/min with a linear gradient of 0.7%/min from 3% Solvent B (100% AcN with 0.1% aqueous formic acid) to 45% B followed by a linear gradient of 1%/min to 80% B. Solvent A was 0.1% aqueous formic acid. The column flow rate was produced by splitting the primary flow rate of 80 L/min from a Surveyor HPLC system (Thermo Finnigan) at a ratio of 1:115.

A NanoSpray Ion Source (Thermo Finnigan) with a 30-m ID fused-silica PicoTip was used to introduce the column effluent into an LCQ Deca ion trap mass spectrometer using the following source conditions:

Capillary Temp: 175 C
Spray Voltage: 1.4kV
Capillary Voltage: 25 V
Tube Lens Offset: 0 V CaM mediates vital processes including inflammation, short-term
memory, the immune response and the cell cycle implicating it in
AIDS, Alzheimers, certain cancers, and other diseases.
Discussion

MS analysis of 900 fmoles of tryptic digest of oxidized CaM revealed 13 peptide fragments that were identified in a single Data Dependent fullscan MS/MS experiment using Dynamic Exclusion (Figure 1). The result from the MS/MS analysis shows coverage for a majority of the amino acid sequence of CaM. In addition to the expected native tryptic fragments, three peaks were identified as peptides containing MetSO (Table 1). Since sulfoxidation of Met residues in CaM plays a crucial role in the activation of target enzymes, the exact position and nature of the oxygen addition (+16 m/z) to the Met residues in the trypti
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