Proteomics of Primary Uveal Melanoma: Insights into Metastasis and Protein Biomarkers

Uveal melanoma metastases are lethal and remain incurable. A quantitative proteomic analysis of 53 metastasizing and 47 non-metastasizing primary uveal melanoma (pUM) was pursued for insights into UM metastasis and protein biomarkers. The metastatic status of the pUM specimens was defined based on clinical data, survival histories, prognostic analyses, and liver histopathology. LC MS/MS iTRAQ technology, the Mascot search engine, and the UniProt human database were used to identify and quantify pUM proteins relative to the normal choroid excised from UM donor eyes. The determined proteomes of all 100 tumors were very similar, encompassing a total of 3935 pUM proteins. Proteins differentially expressed (DE) between metastasizing and non-metastasizing pUM (n = 402) were employed in bioinformatic analyses that predicted significant differences in the immune system between metastasizing and non-metastasizing pUM. The immune proteins (n = 778) identified in this study support the immune-suppressive nature and low abundance of immune checkpoint regulators in pUM, and suggest CDH1, HLA-DPA1, and several DE immune kinases and phosphatases as possible candidates for immune therapy checkpoint blockade. Prediction modeling identified 32 proteins capable of predicting metastasizing versus non-metastasizing pUM with 93% discriminatory accuracy, supporting the potential for protein-based prognostic methods for detecting UM metastasis.

Quantitative proteomic comparison of myofibroblasts derived from bone marrow and cornea

Myofibroblasts are fibroblastic cells that function in wound healing, tissue repair and fibrosis, and arise from bone marrow (BM)-derived fibrocytes and a variety of local progenitor cells. In the cornea, myofibroblasts are derived primarily from stromal keratocytes and from BM-derived fibrocytes after epithelial-stromal and endothelial-stromal injuries. Quantitative proteomic comparison of mature alpha-smooth muscle actin (α-SMA)+ myofibroblasts (verified by immunocytochemistry for vimentin, α-SMA, desmin, and vinculin) generated from rabbit corneal fibroblasts treated with transforming growth factor (TGF) beta-1 or generated directly from cultured BM treated with TGF beta-1 was pursued for insights into possible functional differences. Paired cornea-derived and BM-derived α-SMA+ myofibroblast primary cultures were generated from four New Zealand white rabbits and confirmed to be myofibroblasts by immunocytochemistry. Paired cornea- and BM-derived myofibroblast specimens from each rabbit were analyzed by LC MS/MS iTRAQ technology using an Orbitrap Fusion Lumos Tribrid mass spectrometer, the Mascot search engine, the weighted average quantification method and the UniProt rabbit and human databases. From 2329 proteins quantified with ≥ 2 unique peptides from ≥ 3 rabbits, a total of 673 differentially expressed (DE) proteins were identified. Bioinformatic analysis of DE proteins with Ingenuity Pathway Analysis implicate progenitor-dependent functional differences in myofibroblasts that could impact tissue development. Our results suggest BM-derived myofibroblasts may be more prone to the formation of excessive cellular and extracellular material that are characteristic of fibrosis.

A Comparative Protein Profile of Virgin and Mated Male of Leucinodes Orbonalis Accessory Gland

Male accessory gland (MAG) proteins that are secretory in nature delivered to females, along with sperms affects female reproductive physiology and behavior. In our study, proteomic approaches were employed to identify the MAG proteins of L. orbonalis, a monophagous and most destructive pest on brinjal. A set of 117 spots in virgin MAG and 186 in mated MAG were obtained from 2-D gel electrophoresis. The differentially expressed MAG proteins after mating in comparison with the virgin male were 14 upregulated and 16 down-regulated. We have used MALDI- MS to identify the 13 unique proteins within the virgin MAG of the L. orbonalis and analyzed with the Swiss-Prot database using a Mascot search engine. The proteins were identified as proteolysis regulators, lipid transporter, olfactory protein, metabolism, DNA binding, and hexamerins. This is the first report on proteome analysis of MAG of the Leucinodes orbonalis.

STUDY OF ANTIMICROBIAL PROTEIN FROM THE HEMOLYMPH OF FRESHWATER CRAB OZIOTELPHUSA SENEX SENEX AND ITS EFFICACY AGAINST THE HUMAN PATHOGENS

Objective: The study was done to isolate the antimicrobial protein from the freshwater crab.Methods: Antimicrobial protein was purified by sequential step of ammonium sulfate precipitation, dialysis, ion exchange chromatography, and fast protein liquid chromatography. The apparent molecular mass was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS/MS). Primary structure analysis was done from MASCOT search engine. The antimicrobial activity of the protein was done using well diffusion method against Gram-positive and Gram-negative bacteria.Results: The molecular mass of antimicrobial protein was determined to be 33KDa by SDS-PAGE and MALDI-MS/MS. The antimicrobial protein contains eight peptides which were determined by MASCOT search engine. The protein exhibited antimicrobial activity both for Gram-positive and Gram-negative bacteria.Conclusion: The results could provide information for investigating the antimicrobial protein derived from the hemolymph of the freshwater crab Oziotelphusa senex senex.

Anti-60S ribosomal protein L29 antibody: New anticancer agent discovered from human sera.

3071 Background: Incidence of hepatocellular carcinoma (HCC) is lower in autoimmune hepatitis (AIH) than chronic viral hepatitis. In AIH, serum immunoglobulin G (IgG) levels are associated with the clinical features. In this study, we searched IgG showing anti-tumor effect in sera of AIH patients. Methods: Total IgG was extracted from sera of AIH patients by using protein G. Anti-tumor effects of the total IgG were evaluated by MTT assay using human HCC Huh7 cells and PLC/PRF/5 cells. Autoantigens in membrane proteins of Huh7 cells were screened by immunoprecipitation followed by liquid chromatography-mass spectrometry (LC-MS) shotgun analysis. Results: In one AIH patient without any cancers, addition of total IgG extracted from her serum to the culture inhibited the proliferation of Huh7 cells and PLC/PRF/5 cells, and decreased intracellular levels of β-Catenin and Cyclin-D1. In this patient, autoantigens in membrane proteins were screened, and 60S ribosomal protein L29 (RPL29) was identified from the MS/MS spectra and the SwissProt database using the Mascot Search engine. RPL29 expression in human HCC cell lines including Huh7, PLC/PRF/5, Hep3B, HepG2, HLE, HLF and SK-Hep-1 were identified by Western blot. Next, in the other 25 AIH patients, we investigated the correlation between serum anti-RPL29 levels by indirect ELISA using recombinant RPL29 and anti-tumor effects of total IgG extracted from their sera. Serum anti-RPL29 levels were significantly correlated with anti-tumor effects of total IgG extracted from their sera (P <0.0001). On the other hand, addition of recombinant RPL29 to the culture canceled anti-tumor effects of total IgG extracted from sera of AIH patients showing higher serum anti-RPL29 levels. Additionally, these anti-tumor effects of total IgG extracted from sera of AIH patients were shown by MTT assay using human pancreatic cancer AsPC-1 cells and Panc-1 cells. Conclusions: Anti-RPL29 antibodies showing anti-tumor effect were discovered from human sera. Anti-RPL29 inhibits cancer cell proliferation via down-regulation of Wnt/β-Catenin signaling pathway. Serum anti-RPL29 may be one of immune systems by which the human does not develop cancers.

Standardization and characterization of antigens for the diagnosis of aspergillosis

The aim of this study was to develop and characterize antigens for the diagnosis of aspergillosis. Nine strains of Aspergillus species Aspergillus fumigatus , Aspergillus flavus , and Aspergillus niger were grown in Sabouraud and Smith broth to produce exoantigens. The antigens were tested by immunodiffusion against sera from patients with aspergillosis and other systemic mycoses. The protein fraction of the antigens was detected by SDS–PAGE; Western blot and representative bands were assessed by mass spectrometry coupled to a nano Acquity UltraPerformance LC and analyzed by the Mascot search engine. Concurrently, all sera were tested with Platelia Aspergillus EIA. The most reactive antigens to sera from patients infected by A. fumigatus were produced by A. fumigatus MG2 Sabouraud and pooled A. fumigatus Sabouraud samples, both with a sensitivity of 93% and specificity of 100% and 97%, respectively. Aspergillus niger and A. flavus antigens were reactive against A. niger and A. flavus sera, each one with a sensitivity and specificity of 100%. Two proteins, probably responsible for antigenic activity, β-glucosidase in A. fumigatus and α-amylase in A. niger were attained. The commercial kit had a specificity of 22%, sensitivity of 100%, positive predictive value of 48%, and negative predictive value of 100%. The antigens produced showed high sensitivity and specificity and can be exploited for diagnostics of aspergilloma.

Exhaled Endogenous Particles Contain Lung Proteins

BACKGROUND We recently developed a novel, noninvasive method for sampling nonvolatile material from the distal airways. The method is based on the collection of endogenous particles in exhaled air (PEx). The aim of this study was to characterize the protein composition of PEx and to verify that the origin of PEx is respiratory tract lining fluid (RTLF). METHOD Healthy individuals exhaled into the sampling device, which collected PEx onto a silicon plate inside a 3-stage impactor. After their extraction from the plates, PEx proteins were separated by SDS-PAGE and then analyzed by LC-MS. Proteins were identified by searching the International Protein Index human database with the Mascot search engine. RESULTS Analysis of the pooled samples identified 124 proteins. A comparison of the identified PEx proteins with published bronchoalveolar lavage (BAL) proteomic data showed a high degree of overlap, with 103 (83%) of the PEx proteins having previously been detected in BAL. The relative abundances of the proteins were estimated according to the Mascot exponentially modified protein abundance index protocol and were in agreement with the expected protein composition of RTLF. No amylase was detected, indicating the absence of saliva protein contamination with our sampling technique. CONCLUSIONS Our data strongly support that PEx originate from RTLF and reflect the composition of undiluted RTLF.