Identification Of Protein and Post Translational Modification Markers Of Pulmonary Vasculopathy In Sickle Cell Disease

Pulmonary hypertension (PH), an independent risk factor for mortality in sickle cell disease (SCD), has epidemiologic links to systemic complications such as leg ulcers, priapism and renal disease suggestive of a diffuse vasculopathy. The pathogenesis of SCD related vasculopathy is unclear but altered redox biology is thought to be an important disease modifier. One potential mechanism by which this occurs is via plasma protein post-translational modifications (PTMs). Here we aim to identify a plasma protein and PTM signature profile reflective of PH and pulmonary vasculopathy related to SCD. By targeting both protein and PTM changes, we hoped to enhance the specificity of detecting biomarkers. To control for sex-related protein changes, we limited the present study to females. We obtained peripheral blood samples from 1) SCD patients with a normal echocardiogram (n=10); 2) SCD patients with PH diagnosed by right heart catheterization (n=6) or a tricuspid regurgitant jet velocity > 2.5 m/sec suggestive of pulmonary vasculopathy (n=4) (total n=10); 3) Age- and racially- matched HbAA volunteers (n=11). Plasma was depleted of high abundance proteins using accepted protocols. We performed differential proteomic analysis of plasma and abundant protein-depleted plasma, and differential analysis of PTMs on abundant plasma proteins. Plasma was subjected to trypsin digestion and the peptides were analyzed by differential label-free LCMS/MS. Peptide and protein identification was performed utilizing Proteome Discoverer (Thermo-Fisher) and Mascot (Matrix Science). Data analysis was conducted using the Trans-Proteomics-Pipeline (ISB), Scaffold (Proteome Software), Progenesis LCMS (Nonlinear Dynamics) and STRAP-PTM (in-house). LCMS/MS analysis of the entire plasma proteome revealed more than 2,500 changes in peptide expression between groups and almost 400 peptides which contained PTMs differentiating the SCD patients from the normal volunteers (ANOVA p< 0.05). Analyses indicated significant fold changes for a large group of known plasma protein markers associated with cardiovascular disease, redox biology and metabolism, and the inflammatory host response including alpha-2-HS glycoproteins, ceruloplasmin, apolipoprotein E, transthyretin, kininogen, carbonic anhydrase, angiotensinogen, and complement C4. We also observed changes in the PTMs of several plasma proteins including human albumin, particularly S-cysteinylation on Cys-34, and significant modifications by lipid peroxidation and glycation-end products, such as malonyldialdehyde, 4-hydroxynonenal, and hexose. Further refinement of the data to look only at the SCD samples demonstrated an increased frequency of oxidative PTMs on proteins and peptides from those with PH or an elevated TRV. Additionally, we identified 19 peptides which were distinct in the PH group compared with SCD patients with normal cardiopulmonary function. The corresponding proteins were identified as those important in redox biology, nitric oxide and nitrite signaling, and vasodilatation. This suggests that utilization of proteomics technologies may help to identify unique biomarkers reflective of disease progression and pathogenesis which may help to risk stratify patients and assess response to therapy.