Bivalirudin decreases NO bioavailability by vascular immobilization of myeloperoxidase

Data Supplement

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• Data Supplement -
  Supplemental Figure 1. Mass spectrometric analysis of unmodified and methylated bivalirudin.
  (a) Molecular structure of bivalirudin and fragmentation sites by collision induced dissociation employing a collision energy of 60 V for masses between 116 and 716 Da and a collision energy of 53 V for masses between 711 and 1600 V.
  (b) MS/MS spectrum of unmodified bivalirudin corresponding to the fragmentation given in panel (a).
  (c) Ion chromatogram of bivalirudin as assessed by ESI-LC-MS (m/z 1090.5/356.3). Unmodified bivalirudin eluted after 3.56 minutes using 0.1% formic acid and a linear gradient of acetonitrile + 0.1 % formic acid (5 to 45 % in 7.5 minutes).
  (d) After treatment with BF3-methanol bivalirudin was found to be methylated on the C-terminal end and the glutamic acid side chains (methylation sites bold and in squares).
  (e) The MS/MS spectrum of methylated bivalirudin revealed fragmentation on the same sites to be similar to fragmentation sites of unmodified bivalirudin. The resulting masses of the fragments are shifted by multiples of 14 (mass of CH3: 15 Da, loss of H+ from carboxyl group during reaction) depending on fragmentation site.
  (f) ESI-LC-MS analysis of methylated bivalirudin (m/z 1125.50/384.4) revealed an elution peak after 4.61 minutes using the same linear gradient as for unmodified bivalirudin.

  Supplemental Figure 2. Standard curve for bivalirudin. MRM analysis (m/z 1090.5/356.3) revealed a linear standard curve from 0.01 mg/ml to 20 mg/mL.