Analysis of protein composition of rabbit aqueous humor following two different cataract surgery incision procedures using 2-DE and LC-MS/MS

<p>Abstract</p> <p>Background</p> <p>The aqueous humor (AH), a liquid of the anterior and posterior chamber of the eye, comprises many proteins with various roles and important biological functions. Many of these proteins have not been identified yet and their functions in AH are still unknown. Recently, our laboratory published the protein database of AH obtained from healthy rabbits which expanded known protein identifications by 65%. Our present study extends our previous work and analyses AH following two types of cataract surgery incision procedures (clear corneal and limbal incisions) by using two dimensional gel electrophoresis (2-DE) and liquid chromatography tandem mass spectrometry (LC-MS/MS). Although both incision protocols are commonly used during cataract surgeries, the difference in protein composition and their release into AH following each surgery has never been systematically compared and remains unclear. The first step, which is the focus of this work, is to assess the scale of the protein change, at which time does maximum release occurs and when possible, to identify protein changes.</p> <p>Results</p> <p>Samples of AH obtained prior to surgery and at different time points (0.5, 2, 12, 24 and 48 hours) following surgery (n = 3/protocol) underwent protein concentration determination, 2-DE and LC-MS/MS. There was a large (9.7 to 31.2 mg/mL) and rapid (~0.5 hour) influx of proteins into AH following either incision with a return to baseline quantities after 12 hours and 24 hours for clear corneal and limbal incision, respectively. We identified 80 non-redundant proteins, and compared to our previous study on healthy AH, 67.5% of proteins were found to be surgery-specific. In addition, 51% of those proteins have been found either in clear corneal (20%) or limbal incision (31%) samples.</p> <p>Conclusions</p> <p>Our results imply that a mechanism of protein release into AH after surgery is a global response to the surgery rather than increase in amount of protective proteins found in healthy AH and a mechanism of protein release for each type of incision procedure could be different. Although the total protein concentration was increased (at 0.5 and 2 hour time points and between types of surgery) many of 2-DE protein spots were similar based on 2-DE and MS analyses, and only a small number of protein spots changed with either the time points or surgical conditions (0.4 -1.9%). This suggests that the high protein content is due to an increase in the concentration of the same proteins with only a few unique proteins being altered per time point and with the different surgery type. This is the first report on the comparison of AH protein composition following two different cataract surgery procedures and it establishes the basis for better understanding of protein release into AH during events such as cataract surgery or other possible intervention to the eyes.</p

Proteome Analysis of Liver Cells Expressing a Full-Length Hepatitis C Virus (HCV) Replicon and Biopsy Specimens of Posttransplantation Liver from HCV-Infected Patients

The development of a reproducible model system for the study of hepatitis C virus (HCV) infection has the potential to significantly enhance the study of virus-host interactions and provide future direction for modeling the pathogenesis of HCV. While there are studies describing global gene expression changes associated with HCV infection, changes in the proteome have not been characterized. We report the first large-scale proteome analysis of the highly permissive Huh-7.5 cell line containing a full-length HCV replicon. We detected >4,200 proteins in this cell line, including HCV replicon proteins, using multidimensional liquid chromatographic (LC) separations coupled to mass spectrometry. Consistent with the literature, a comparison of HCV replicon-positive and -negative Huh-7.5 cells identified expression changes of proteins involved in lipid metabolism. We extended these analyses to liver biopsy material from HCV-infected patients where a total of >1,500 proteins were detected from only 2 μg of liver biopsy protein digest using the Huh-7.5 protein database and the accurate mass and time tag strategy. These findings demonstrate the utility of multidimensional proteome analysis of the HCV replicon model system for assisting in the determination of proteins/pathways affected by HCV infection. Our ability to extend these analyses to the highly complex proteome of small liver biopsies with limiting protein yields offers the unique opportunity to begin evaluating the clinical significance of protein expression changes associated with HCV infection

Proteomics of mouse heart ventricles reveals mitochondria and metabolism as major targets of a post-infarction short-acting glp1ra-therapy

Cardiovascular disease is the main cause of death worldwide, making it crucial to search for new therapies to mitigate major adverse cardiac events (MACEs) after a cardiac ischemic episode. Drugs in the class of the glucagon-like peptide-1 receptor agonists (GLP1Ra) have demonstrated benefits for heart function and reduced the incidence of MACE in patients with diabetes. Previously, we demonstrated that a short-acting GLP1Ra known as DMB (2-quinoxalinamine, 6,7-dichloro-N-[1,1-dimethylethyl]-3-[methylsulfonyl]-,6,7-dichloro-2-methylsulfonyl-3-N-tert-butylaminoquinoxaline or compound 2, Sigma) also mitigates adverse postinfarction left ventricular remodeling and cardiac dysfunction in lean mice through activation of parkin-mediated mitophagy following infarction. Here, we combined proteomics with in silico analysis to characterize the range of effects of DMB in vivo throughout the course of early postinfarction remodeling. We demonstrate that the mitochondrion is a key target of DMB and mitochondrial respiration, oxidative phosphorylation and metabolic processes such as glycolysis and fatty acid beta-oxidation are the main biological processes being regulated by this compound in the heart. Moreover, the overexpression of proteins with hub properties identified by protein–protein interaction networks, such as Atp2a2, may also be important to the mechanism of action of DMB. Data are available via ProteomeXchange with identifier PXD027867