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

Background: 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 (PTMs) occur on plasma proteins and are important in disease pathogenesis. We previously reported the identification of one PTM, a malondialdehyde adduct on peptide 146–159 of albumin and sought to do a more extensive evaluation of this protein to determine the presence of other abnormalities.

Methods: 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 spectrometry (MS) and liquid chromatography (LC)-MS/MS tandem mass spectrometry. Proteomic analyses were performed on all samples and post-translational modifications characterized by MS/MS.

Results: We have characterized several additional peptides of albumin from patients with PH of SCA that bear lipid peroxidation and glycation adducts. Our comprehensive LC-MS/MS results have allowed us to identify 4-hydroxynonenal (HNE), hexose (HEX) and malonyl adduction at numerous, distinct cysteine, histidine, lysine, and threonine albumin residues.

Conclusion: Increased oxidant burden and altered redox biology is characteristic of PH in SCA. Plasma proteins, such as albumin, are a target for oxidants and changes in their structure may play a role in disease pathogenesis.