This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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 is being obtained from a cohort ot 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) are used to separate plasma samples into albumin-enriched and albumin-deplete fractions. The degree of plasma separation is confirmed by SDS-PAGE chromatography. After cleavage with trypsin, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) and LC/MS are performed for peptide analysis of the albumin-enriched fractions. The albumin-depleted fractions are analyzed by two-dimensional protein fractionation (PF2D), followed by MALDI MS and LC/MS, as well as 2D-SDS PAGE. Numerous adducts have been found in albumin fractions from patients. The occurrence of these modifications is being evaluated in further samples, using the STRAP-PTM softwarre written in-house.
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