This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.The vascular pathology of sickle cell disease (SCD) is characterized by altered nitric oxide (NO) metabolism and increased oxidant stress. The etiology of pulmonary hypertension (PH), an increasingly recognized complication of SCD, is, however, unclear and likely multi-factorial. We hypothesize that, in PH of SCD, oxidative stress results in post-translational protein modifications (PTMs) such as tyrosine nitrosylation that contribute to disease pathogenesis. To identify oxidative targets within a high abundance protein, albumin, and lower abundance proteins, we utilized a proteomic approach. Platelet-poor plasma was obtained from four age, gender and racially matched subjects in each of the following groups: 1) Sickle cell anemia (Hb-SS) with PH; 2) Hb-SS without PH; 3) Normal hemoglobin controls (Hb-AA) with PH; 4) Hb-AA volunteers without cardiopulmonary disease. Albumin removal cartridges (EMD Biosciences) were used to separate plasma samples into albumin-enriched and albumin-deplete fractions. The degree of plasma separation was confirmed by SDS-PAGE chromatography. After cleavage with trypsin, matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) was performed for peptide analysis of the albumin-enriched fractions. Results were correlated with those obtained by liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS). The albumin-depleted fractions were analyzed by two-dimensional protein fractionation (PF2D), followed by MALDI MS. An adduct of malonaldehyde was found in albumin fractions from patients with PH. The occurrence of this modification will be evaluated in further samples. A manuscript describing the preliminary results has been submitted.
Showing the most recent 10 out of 253 publications
