Accurate determination of protein methionine oxidation by stable isotope labeling and LC-MS analysis

Transcription

1 Accurate determination of protein methionine oxidation by stable isotope labeling and LC-MS analysis Hongcheng Liu*, Gomathinayagam Ponniah, Alyssa Neil, Rekha Patel, Bruce Andrien Protein Characterization, Alexion Pharmaceuticals Inc, 352 Knotter Drive, Cheshire, CT06410 *Corresponding author: Hongcheng Liu Telephone:

2 Oxidation of peptide 2 Extracted ion chromatograms of peptide 2 from searching m/z of the triply charged ions based on the molecular weights of peptide 2 containing either Met or Met sulfoxide are shown in supplemental Figure 1. The observed molecular weight (MH + ) of the peak eluted at the retention time of approximately 58 minutes is , which is in good agreement with the calculated molecular weight of The observed molecular weight of the peak eluted at the retention time of approximately 51 minutes is , which is 16 Da higher than that of the peak eluted at the retention time of 57 minutes. This molecular weight is in good agreement with the molecular weight of the peptide containing Met sulfoxide. The peptide containing Met sulfoxide eluted earlier than the peptide containing Met due to increased polarity. Supplemental Figure 1. EIC of peptide 2 containing either Met (A) or Met sulfoxide (B). Mass spectra of the corresponding peptides are shown as insets as triply charged ions. Other peaks shown in the EIC containing peptides with m/z range similar to peptide 2, but are not related to peptide 2.

3 Procedure to calculate the percentage of Met sulfoxide originally in the sample The multiple step calculation procedure will be as following, Step 1. Calculation of the overlap of the third peak of m/z to the peak of m/z The m/z peak distribution of peptide 1without oxidation is shown in supplemental Figure 2. The peak ratio of the third peak (m/z 837.5) over the first peak (835.5) was calculated to be This ratio was used for calculation of the overlap because the isotope peak distributions of peptide 1 with or without oxidation are similar. Supplemental Figure 2. Isotope peak distribution of peptide 1 containing Met The overlap of the third peak of the m/z peak series to the peak intensity at m/z (Supplemental Figure 3) was calculated by multiplying the peak intensity at m/z by This overlap needs to be subtracted from the peak intensity of m/z Supplemental Figure 3. Mass spectrum of peptide 1 containing Met sulfoxide with either 16 O-atom or 18 O-atom.

4 Step 2. Calculation of the contribution of the presence of low level of 16 O-hydrogen peroxide in the 18 O- hydrogen peroxide reagent Mass spectrum of a synthesized peptide with the same amino acid sequence as peptide 1 after oxidation using 18 O-hydrogen peroxide is shown in supplemental Figure 4. The peak of m/z represents oxidation by 16 O-hydrogen peroxide. The peak of m/z represents oxidation by 18 O-hydrogen peroxide, ignoring the overlap of the third peak of the m/z series because of its extremely low intensity. The ratio of m/z over m/z of is from triplicate analyses. Therefore, the contribution of the low percentage of 16 O in the 18 O-hydrogen peroxide was calculated by multiplying by the peak intensity at m/z after subtraction of the overlap calculated from step 1. The initial oxidation level of this synthesized peptide was only 0.2%, it was also not considered in the calculation. Supplemental Figure 4. Peak distribution of the synthesized peptide oxidized by 18 O-hydrogen peroxide. Step 3. Calculation of the percentage of Met sulfoxide originally in the sample Assuming, A=the peak intensity of m/z corresponding to Met sulfoxide originally in the sample B=the peak intensity of m/z corresponding to Met sulfoxide generated during sample preparation The percentage of Met sulfoxide=a/(a+b)

5 A=the peak intensity at m/z after subtraction of the contribution of the low level of 16 O-hydrogen peroxide in the 18 O-hydrogen peroxide reagent B=the peak intensity at m/z after subtraction of the contribution of the third peak of the m/z peak series