Identification of Oxidative Post-Translational Modifications on Plasma Albumin in Patients with Pulmonary Hypertension of Sickle Cell Anemia.

Pulmonary hypertension (PH) in sickle cell anemia (SCA) is characterized by decreased nitric oxide bioavailability; which may, in part, be related to increased oxidative stress. It is possible that via protein post-translational modifications, oxidants are able to affect both protein structure and function. We hypothesized that, in patients with SCA and PH, oxidative post-translational modifications occur on plasma proteins and are important in disease pathogenesis. To examine this hypothesis, we chose to determine if oxidative post-translational modifications occur in a large-abundance protein, albumin, as a reflection of the presence of more widespread protein oxidative damage. Plasma was obtained from subjects with:

1. SCA and PH (n=5);

2. SCA steady-state without PH (n=4);

3. Pulmonary Arterial Hypertension (PAH) (n=4);

4. no evidence of cardiopulmonary disease (n=4).

Platelet-poor plasma was separated into albumin-enriched and albumin-depleted fractions. The albumin-enriched fraction was subjected to proteolytic digestion by trypsin and studied by matrix assisted laser desorption ionization (MALDI) mass spectroscopy (MS) and liquid chromatography (LC)-MS/MS tandem mass spectrometry. Proteomics analyses were performed on all samples and post-translational modifications characterized by MS/MS. Results were confirmed by Western and dot-blot analysis using commercially available antibodies. In the albumin fraction, we identified peaks of differential post-translational modification on albumin peptide 146–159 only in PAH and SCA and PH samples; this PTM was identified to be a malondialdehyde adduct. The presence of a malondialdehyde adduct on albumin was confirmed by LC-MS/MS and Western analysis. We have identified a malondialdehyde adduct in plasma albumin that appears to be a common link between PH related to SCA and PAH. This finding supports the notion that oxidative stress modulates the pathogenesis of PH of SCA and suggests that this and other post-translational modifications may be important biomarkers of disease.