Characterizing the normal proteome of human ciliary body

Background

The ciliary body is the circumferential muscular tissue located just behind the iris in the anterior chamber of the eye. It plays a pivotal role in the production of aqueous humor, maintenance of the lens zonules and accommodation by changing the shape of the crystalline lens. The ciliary body is the major target of drugs against glaucoma as its inhibition leads to a drop in intraocular pressure. A molecular study of the ciliary body could provide a better understanding about the pathophysiological processes that occur in glaucoma. Thus far, no large-scale proteomic investigation has been reported for the human ciliary body.

Results

In this study, we have carried out an in-depth LC-MS/MS-based proteomic analysis of normal human ciliary body and have identified 2,815 proteins. We identified a number of proteins that were previously not described in the ciliary body including importin 5 (IPO5), atlastin-2 (ATL2), B-cell receptor associated protein 29 (BCAP29), basigin (BSG), calpain-1 (CAPN1), copine 6 (CPNE6), fibulin 1 (FBLN1) and galectin 1 (LGALS1). We compared the plasma proteome with the ciliary body proteome and found that the large majority of proteins in the ciliary body were also detectable in the plasma while 896 proteins were unique to the ciliary body. We also classified proteins using pathway enrichment analysis and found most of proteins associated with ubiquitin pathway, EIF2 signaling, glycolysis and gluconeogenesis.

Conclusions

More than 95% of the identified proteins have not been previously described in the ciliary body proteome. This is the largest catalogue of proteins reported thus far in the ciliary body that should provide new insights into our understanding of the factors involved in maintaining the secretion of aqueous humor. The identification of these proteins will aid in understanding various eye diseases of the anterior segment such as glaucoma and presbyopia.     

Canonical labeling of proteome maps

We propose a canonical labeling of proteome maps, which enables one to sort and catalog the maps in a simple way. The canonical label of a proteome map is based on the canonical labeling of vertexes of Hasse diagram embedded in the map resulting in the adjacency matrix, the rows of which when viewed as binary numbers are the smallest possible such numbers. The use of the approach in documentation is illustrated with the proteome maps of liver cells of healthy male Fisher F344 rats and the rats treated with different peroxisome proliferators.     

Peptidomics technologies for human body fluids

Peptides play a central role in many physiological processes. In order to analyse comprehensively all peptides and small proteins of a whole organism or a subsystem (peptidome), the use of technologies other than 2D gel electrophoresis is necessary. Approaches that use liquid chromatography or affinity purification and mass spectrometric identification have now been developed and applied successfully to the analysis of human body fluids.     

Peptidomics technologies for human body fluids

Peptides play a central role in many physiological processes. In order to analyse comprehensively all peptides and small proteins of a whole organism or a subsystem (peptidome), the use of technologies other than 2D gel electrophoresis is necessary. Approaches that use liquid chromatography or affinity purification and mass spectrometric identification have now been developed and applied successfully to the analysis of human body fluids.     

Two-dimensional maps and databases of the human macrophage proteome and secretome

Macrophages exert a crucial, but still incompletely known, role in complex disorders such inflammatory, immunological, and infectious diseases. A differential proteomic approach should help to elucidate the macrophage dysfunctions involved in these diseases. With this goal in mind, we established the first two-dimensional maps of the human macrophage proteome and secretome. Intracellular and secreted proteins were extracted from monocyte-derived macrophages obtained from healthy donors (n = 16), and separated by two-dimensional gel electrophoresis. Silver-stained gels were analyzed using Progenesis software. A high level of between-gel reproducibility was obtained, allowing us to generate two patterns specific of the macrophage proteome and secretome, respectively. A total of 127 and 66 distinct intracellular and secreted polypeptide spots, corresponding to 100 and 38 different proteins, respectively, were identified by matrix assisted laser desorption/ionisation-mass spectrometry. The two-dimensional reference maps and databases resulting from this study confirm that macrophages are involved in a wide range of biological functions, and that they provide a useful tool for a wide array of investigators involved in macrophage biology, allowing to investigate the macrophage protein changes associated with various disorders or environmental stimuli.     

High resolution proteome/peptidome analysis of body fluids by capillary electrophoresis coupled with MS

All organisms contain thousands of proteins and peptides in their body fluids. A deeper insight into the functional relevance of these polypeptides under different physiological and pathophysiological conditions and the discovery of specific peptide biomarkers would greatly enhance both diagnosis and therapy of specific diseases. Proteomic methods can provide means to accomplish this grand medical vision. In this review, we will focus on the potential use of proteome analysis for clinical applications, such as disease diagnosis and assessment of response to therapy. We focus on CE coupled with MS (CE-MS) and review in detail different aspects of CE-MS coupling and the results obtained using CE-MS analysis of clinically relevant samples. We also discuss clinical applications of the technology for the diagnosis of renal diseases, urogenital cancer, and arteriosclerosis as well as monitoring the responses to therapeutic interventions.     

Size heterogeneity of epidermal growth factor in human body fluids

We measured the concentration of immunoreactive (IR) hEGF in various body fluids by radioimmunoassay (RIA) and evaluated its size heterogeneity by size exclusion high performance liquid chromatography combined with RIA or with time-resolved immunoflurometric assay (TR-IFMA). Mean concentration was 80 ng/ml in urine, 65 ng/ml in milk, 50 ng/ml in seminal plasma, 25 ng/ml in armpit sweat, 1 ng/ml in breast sweat, 0.3 ng/ml in third-trimester amniotic fluid, 3 ng/ml in saliva, 1.5 ng/ml in tears and 0.3 ng/ml in gastric juice.

All the fluids except armpit sweat and gastric juice contained two to five molecular sizes of IR-hEGF. As well as the 6200-dalton (6.2kDa) hEGF we found at least four other different molecular sizes with approximate weights of 300, 150, 70 and 20kDa. The authentic 6.2kDa form made up >90% of the total IR-hEGF in all except the amniotic fluid where its proportion was 71%, and the seminal plasma where the proportion could not be determined.     

Human body fluid proteome analysis

The focus of this article is to review the recent advances in proteome analysis of human body fluids, including plasma/serum, urine, cerebrospinal fluid, saliva, bronchoalveolar lavage fluid, synovial fluid, nipple aspirate fluid, tear fluid, and amniotic fluid, as well as its applications to human disease biomarker discovery. We aim to summarize the proteomics technologies currently used for global identification and quantification of body fluid proteins, and elaborate the putative biomarkers discovered for a variety of human diseases through human body fluid proteome (HBFP) analysis. Some critical concerns and perspectives in this emerging field are also discussed. With the advances made in proteomics technologies, the impact of HBFP analysis in the search for clinically relevant disease biomarkers would be realized in the future.     

Generating and navigating proteome maps using mass spectrometry

Proteomes, the ensembles of all proteins expressed by cells or tissues, are typically analysed by mass spectrometry. Recent technical and computational advances have greatly increased the fraction of a proteome that can be identified and quantified in a single study. Current mass spectrometry-based proteomic strategies have the potential to reproducibly, accurately, quantitatively and comprehensively measure any protein or whole proteomes from cells and tissues at different states. Achieving these goals will require complete proteome maps and analytical strategies that use these maps as prior information and will greatly enhance the impact of proteomics on biological and clinical research.     

Inter-alpha-trypsin inhibitor-related immunoreactivity in human tissues and body fluids.

The distribution of inter-alpha-trypsin inhibitor (ITI) and related inhibitors was investigated in normal human tissues and body fluids by using an enzyme-linked immunosorbent assay (ELISA) and a streptavidin-biotin-peroxidase immunohistochemical technique. ITI-related immunoreactivity was localized in different cell types of various organs, such as liver, kidney, testis, gross intestine, cutis and brain. Specific immunoreactivity was also detected in serum, urine and bronchial mucus. This widespread, but not ubiquitous pattern of localization suggests that, in addition to the well known plasmatic role, ITI and/or ITI-related inhibitors may play a number of different physiological roles in various human tissues.     

Two‐dimensional proteome reference maps for the human pathogenic filamentous fungus Aspergillus fumigatus

The filamentous fungus Aspergillus fumigatus has become the most important airborne fungal pathogen causing life‐threatening infections in immunosuppressed patients. We established a 2‐D reference map for A. fumigatus. Using MALDI‐TOF‐MS/MS, we identified 381 spots representing 334 proteins. Proteins involved in cellular metabolism, protein synthesis, transport processes and cell cycle were most abundant. Furthermore, we established a protocol for the isolation of mitochondria of A. fumigatus and developed a mitochondrial proteome reference map. 147 proteins represented by 234 spots were identified.     

Normal levels of von Willebrand Factor antigen in human body fluids

von Willebrand Factor antigen is present in serum, plasma, synovial fluid and semen, and absent from other body fluids including breast milk, urine, saliva, and cerebro-spinal fluid. Levels are higher in plasma relative to serum. There is no difference between levels in different sexes, and no increase with age, but levels in cord blood are raised.     

Development of an effective sample preparation method for the proteome analysis of body fluids using 2-D gel electrophoresis

A sample preparation is still the most critical step in two-dimensional electrophoresis (2-DE) and should be optimized for each type of sample. In this study, a protein extraction method from body fluids was developed using a combined centrifugal filter device and a sample treating buffer. When plasma, amniotic fluid, urine, and tear were tested with this method, the recovery of protein reached almost 90% and high-quality separation of 2-DE gel was obtained.