Comprehensive proteomic analysis of human bile

Bile serves diverse functions from metabolism to transport. In addition to acids and salts, bile is composed of proteins secreted or shed by the hepatobiliary system. Although there have been previous efforts to catalog biliary proteins, an in-depth analysis of the bile proteome has not yet been reported. We carried out fractionation of non-cancerous bile samples using a multipronged approach (SDS-PAGE, SCX and OFFGEL) followed by MS analysis on an LTQ-Orbitrap Velos mass spectrometer using high resolution at both MS and MS/MS levels. We identified 2552 proteins - the largest number of proteins reported in human bile till date. To our knowledge, there are no previous studies employing high-resolution MS reporting a more detailed catalog of any body fluid proteome in a single study. We propose that extensive fractionation coupled to high-resolution MS can be used as a standard methodology for in-depth characterization of any body fluid. This catalog should serve as a baseline for the future studies aimed at discovering biomarkers from bile in gallbladder, hepatic, and biliary cancers.     

A proteomic analysis of human bile

We have carried out a comprehensive characterization of human bile to define the bile proteome. Our approach involved fractionation of bile by one-dimensional gel electrophoresis and lectin affinity chromatography followed by liquid chromatography tandem mass spectrometry. Overall, we identified 87 unique proteins, including several novel proteins as well as known proteins whose functions are unknown. A large majority of the identified proteins have not been previously described in bile. Using lectin affinity chromatography and enzymatically labeling of asparagine residues carrying glycan moieties by (18)O, we have identified a total of 33 glycosylation sites. The strategy described in this study should be generally applicable for a detailed proteomic analysis of most body fluids. In combination with "tagging" approaches for differential proteomics, our method could be used for identification of cancer biomarkers from any body fluid.     

Three-dimensional ultrastructure and quantitative analysis of the human Sertoli cell nucleolus

The nucleolus of the human Sertoli cell displays a spontaneous segregation of its components and has only one or 2 large fibrillar centers. The 3-dimensional reconstruction and quantitative analysis of its components was undertaken using a Quantimet 900 image analysis system in order to define the spatial relationships between the dense fibrillar component and the fibrillar center and especially to investigate whether threads of dense fibrillar component exist independently, without being linked to a fibrillar center. Our 3D reconstructions demonstrated that the dense fibrillar threads or sheets were never independent of fibrillar centers. These structures belonged to a continuous network that joined the layer of dense fibrils surrounding the fibrillar center. When the nucleolus contained 2 different-sized fibrillar centers, quantitative analysis showed that there was a proportional relationship between the volume of the dense fibrillar component and the volume of the fibrillar center. These data, compared with those previously obtained by means of autoradiographic techniques, suggest that the rDNA-containing chromatin passes through the fibrillar center and unwinds from there into the dense fibrillar component.     

Targeted tissue proteomic analysis of human astrocytomas

Complicating proteomic analysis of whole tissues is the obvious problem of cell heterogeneity in tissues, which often results in misleading or confusing molecular findings. Thus, the coupling of tissue microdissection for tumor cell enrichment with capillary isotachophoresis-based selective analyte concentration not only serves as a synergistic strategy to characterize low abundance proteins, but it can also be employed to conduct comparative proteomic studies of human astrocytomas. A set of fresh frozen brain biopsies were selectively microdissected to provide an enriched, high quality, and reproducible sample of tumor cells. Despite sharing many common proteins, there are significant differences in the protein expression level among different grades of astrocytomas. A large number of proteins, such as plasma membrane proteins EGFR and Erbb2, are up-regulated in glioblastoma. Besides facilitating the prioritization of follow-on biomarker selection and validation, comparative proteomics involving measurements in changes of pathways are expected to reveal the molecular relationships among different pathological grades of gliomas and potential molecular mechanisms that drive gliomagenesis.     

Comprehensive proteomic analysis of human pancreatic juice

Proteomic technologies provide an excellent means for analysis of body fluids for cataloging protein constituents and identifying biomarkers for early detection of cancers. The biomarkers currently available for pancreatic cancer, such as CA19-9, lack adequate sensitivity and specificity contributing to late diagnosis of this deadly disease. In this study, we carried out a comprehensive characterization of the "pancreatic juice proteome" in patients with pancreatic adenocarcinoma. Pancreatic juice was first fractionated by 1-dimensional gel electrophoresis and subsequently analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). A total of 170 unique proteins were identified including known pancreatic cancer tumor markers (e.g., CEA, MUC1) and proteins overexpressed in pancreatic cancers (e.g., hepatocarcinoma-intestine-pancreas/pancreatitis-associated protein (HIP/PAP) and lipocalin 2). In addition, we identified a number of proteins that have not been previously described in pancreatic juice (e.g., tumor rejection antigen (pg96) and azurocidin). Interestingly, a novel protein that is 85% identical to HIP/PAP was identified, which we have designated as PAP-2. The proteins identified in this study could be directly assessed for their potential as biomarkers for pancreatic cancer by quantitative proteomics methods or immunoassays.     

Comprehensive proteomic analysis of human cervical-vaginal fluid

Cervical-vaginal fluid (CVF) is a potential rich source of biomarkers for enhancing our understanding of human parturition and pathologic conditions affecting pregnancy. In this study, we performed a comprehensive survey of the CVF proteome in pregnancy utilizing multidimensional liquid chromatography (2D-LC) coupled with mass spectrometry and gel-electrophoresis-based protein separation and identification. In total, 150 unique proteins were identified using multiple protein identification algorithms. Metabolism (32%) and immune response-related (22%) proteins are the major functional categories represented in the CVF proteome. A comparison of the CVF, serum, and amniotic fluid proteomes showed that 77 proteins are unique to CVF, while 56 and 17 CVF proteins also occur in serum and amniotic fluid, respectively. This data set provides a foundation for evaluation of these proteins as potential CVF biomarkers for noninvasive diagnosis of pregnancy-related disorders.     

A wiring of the human nucleolus

Recent proteomic efforts have created an extensive inventory of the human nucleolar proteome. However, approximately 30% of the identified proteins lack functional annotation. We present an approach of assigning function to uncharacterized nucleolar proteins by data integration coupled to a machine-learning method. By assembling protein complexes, we present a first draft of the human ribosome biogenesis pathway encompassing 74 proteins and hereby assign function to 49 previously uncharacterized proteins. Moreover, the functional diversity of the nucleolus is underlined by the identification of a number of protein complexes with functions beyond ribosome biogenesis. Finally, we were able to obtain experimental evidence of nucleolar localization of 11 proteins, which were predicted by our platform to be associates of nucleolar complexes. We believe other biological organelles or systems could be "wired" in a similar fashion, integrating different types of data with high-throughput proteomics, followed by a detailed biological analysis and experimental validation.     

Comparative analysis of the population polymorphism of human silver-staining chromosomal nucleolus organizer regions

The population study of nucleus organizing regions (NOR) activity in individuals of Georgian nationality was carried out using the Ag-method. The results obtained were compared with those for other populations: Russian, Viena-Ulm, Estonian etc. The mean number of active NORs for Georgians is 9.0, which significantly exceeds analogous indexes for the rest of populations, except Viena-Ulm one. The population differences in the distribution of NOR activity and the ability to be stained was found for individual NORs. The lowest activity was registered in chromosome 14, the highest being observed for chromosomes 21 and 13. It is assumed that some mechanism exists that controls the level of general NOR activity in the cell.     

High-throughput proteomic analysis of human infiltrating ductal carcinoma of the breast

Large-scale proteomics will play a critical role in the rapid display, identification and validation of new protein targets, and elucidation of the underlying molecular events that are associated with disease development, progression and severity. However, because the proteome of most organisms are significantly more complex than the genome, the comprehensive analysis of protein expression changes will require an analytical effort beyond the capacity of standard laboratory equipment. We describe the first high-throughput proteomic analysis of human breast infiltrating ductal carcinoma (IDCA) using OCT (optimal cutting temperature) embedded biopsies, two-dimensional difference gel electrophoresis (2-D DIGE) technology and a fully automated spot handling workstation. Total proteins from four breast IDCAs (Stage I, IIA, IIB and IIIA) were individually compared to protein from non-neoplastic tissue obtained from a female donor with no personal or family history of breast cancer. We detected differences in protein abundance that ranged from 14.8% in stage I IDCA versus normal, to 30.6% in stage IIB IDCA versus normal. A total of 524 proteins that showed > or = three-fold difference in abundance between IDCA and normal tissue were picked, processed and identified by mass spectrometry. Out of the proteins picked, approximately 80% were unambiguously assigned identities by matrix-assisted laser desorbtion/ionization-time of flight mass spectrometry or liquid chromatography-tandem mass spectrometry in the first pass. Bioinformatics tools were also used to mine databases to determine if the identified proteins are involved in important pathways and/or interact with other proteins. Gelsolin, vinculin, lumican, alpha-1-antitrypsin, heat shock protein-60, cytokeratin-18, transferrin, enolase-1 and beta-actin, showed differential abundance between IDCA and normal tissue, but the trend was not consistent in all samples. Out of the proteins with database hits, only heat shock protein-70 (more abundant) and peroxiredoxin-2 (less abundant) displayed the same trend in all the IDCAs examined. This preliminary study demonstrates quantitative and qualitative differences in protein abundance between breast IDCAs and reveals 2-D DIGE portraits that may be a reflection of the histological and pathological status of breast IDCA.     

Large-scale and high-confidence proteomic analysis of human seminal plasma

Background  

The development of mass spectrometric (MS) techniques now allows the investigation of very complex protein mixtures ranging from subcellular structures to tissues. Body fluids are also popular targets of proteomic analysis because of their potential for biomarker discovery. Seminal plasma has not yet received much attention from the proteomics community but its characterization could provide a future reference for virtually all studies involving human sperm. The fluid is essential for the survival of spermatozoa and their successful journey through the female reproductive tract.     

System-based proteomic analysis of the interferon response in human liver cells

Background

Interferons (IFNs) play a critical role in the host antiviral defense and are an essential component of current therapies against hepatitis C virus (HCV), a major cause of liver disease worldwide. To examine liver-specific responses to IFN and begin to elucidate the mechanisms of IFN inhibition of virus replication, we performed a global quantitative proteomic analysis in a human hepatoma cell line (Huh7) in the presence and absence of IFN treatment using the isotope-coded affinity tag (ICAT) method and tandem mass spectrometry (MS/MS).

Results

In three subcellular fractions from the Huh7 cells treated with IFN (400 IU/ml, 16 h) or mock-treated, we identified more than 1,364 proteins at a threshold that corresponds to less than 5% false-positive error rate. Among these, 54 were induced by IFN and 24 were repressed by more than two-fold, respectively. These IFN-regulated proteins represented multiple cellular functions including antiviral defense, immune response, cell metabolism, signal transduction, cell growth and cellular organization. To analyze this proteomics dataset, we utilized several systems-biology data-mining tools, including Gene Ontology via the GoMiner program and the Cytoscape bioinformatics platform.

Conclusions

Integration of the quantitative proteomics with global protein interaction data using the Cytoscape platform led to the identification of several novel and liver-specific key regulatory components of the IFN response, which may be important in regulating the interplay between HCV, interferon and the host response to virus infection.     

Head and flagella subcompartmental proteomic analysis of human spermatozoa

Subcellular proteomics not only deepens our knowledge of what proteins are present within cells, but also opens our understanding as to where those proteins reside. Given the highly differentiated, cross‐linked state of spermatozoa, such studies have proven difficult to perform. In this study we have fractionated spermatozoa into two components, consisting of either the head or flagellar region. Following SDS‐PAGE, 1 mm slices were digested and used for LC‐MS/MS analysis. In total, 1429 proteins were identified with 721 proteins being exclusively found in the tail and 521 exclusively in the head. Not only is this the largest reported proteomic analysis of human spermatozoa, but also it has provided novel insights into the compartmentalization of proteins, particularly receptors, never previously reported to be present in this cell type.     

Comparative proteomic analysis of human lung telocytes with fibroblasts

Telocytes (TCs) were recently described as interstitial cells with very long prolongations named telopodes (Tps; www.telocytes.com ). Establishing the TC proteome is a priority to show that TCs are a distinct type of cells. Therefore, we examined the molecular aspects of lung TCs by comparison with fibroblasts (FBs). Proteins extracted from primary cultures of these cells were analysed by automated 2‐dimensional nano‐electrospray ionization liquid chromatography tandem mass spectrometry (2D Nano‐ESI LC‐MS/MS). Differentially expressed proteins were screened by two‐sample t‐test (P < 0.05) and fold change (>2), based on the bioinformatics analysis. We identified hundreds of proteins up‐ or down‐regulated, respectively, in TCs as compared with FBs. TC proteins with known identities are localized in the cytoskeleton (87%) and plasma membrane (13%), while FB up‐regulated proteins are in the cytoskeleton (75%) and destined to extracellular matrix (25%). These identified proteins were classified into different categories based on their molecular functions and biological processes. While the proteins identified in TCs are mainly involved in catalytic activity (43%) and as structural molecular activity (25%), the proteins in FBs are involved in catalytic activity (24%) and in structural molecular activity, particularly synthesis of collagen and other extracellular matrix components (25%). Anyway, our data show that TCs are completely different from FBs. In conclusion, we report here the first extensive identification of proteins from TCs using a quantitative proteomics approach. Protein expression profile shows many up‐regulated proteins e.g. myosin‐14, periplakin, suggesting that TCs might play specific roles in mechanical sensing and mechanochemical conversion task, tissue homoeostasis and remodelling/renewal. Furthermore, up‐regulated proteins matching those found in extracellular vesicles emphasize TCs roles in intercellular signalling and stem cell niche modulation. The novel proteins identified in TCs will be an important resource for further proteomic research and it will possibly allow biomarker identification for TCs. It also creates the premises for understanding the pathogenesis of some lung diseases involving TCs.     

Clonal analysis of the intercellular variability in the functioning of human chromosomal nucleolus organizer regions

Intercellular variability of NOR activity detected with the aid of Ag-staining of human chromosomes was studied in mass and cloned fibroblast cultures obtained from 3 individuals. The intercellular variability was determined by different staining of one of 10 NORs. According to this trait the heterogeneity of the cell population was discovered in all cloned lines, with this heterogeneity being the same as in the parent cultures. That concerned the number of a variable chromosome and the percentage of the cells with Ag-stained and unstained chromosomes. It is suggested that genetic determination in the progenies of the somatic cells concerns the whole spectrum of potential variability observed in cell populations.