| 1. |
- Danielsson, Angelika, et al.
(författare)
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The Human Pancreas Proteome Defined by Transcriptomics and Antibody-Based Profiling
- 2014
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 9:12, s. e115421-
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Tidskriftsartikel (refereegranskat)abstract
- The pancreas is composed of both exocrine glands and intermingled endocrine cells to execute its diverse functions, including enzyme production for digestion of nutrients and hormone secretion for regulation of blood glucose levels. To define the molecular constituents with elevated expression in the human pancreas, we employed a genome-wide RNA sequencing analysis of the human transcriptome to identify genes with elevated expression in the human pancreas. This quantitative transcriptomics data was combined with immunohistochemistry-based protein profiling to allow mapping of the corresponding proteins to different compartments and specific cell types within the pancreas down to the single cell level. Analysis of whole pancreas identified 146 genes with elevated expression levels, of which 47 revealed a particular higher expression as compared to the other analyzed tissue types, thus termed pancreas enriched. Extended analysis of in vitro isolated endocrine islets identified an additional set of 42 genes with elevated expression in these specialized cells. Although only 0.7% of all genes showed an elevated expression level in the pancreas, this fraction of transcripts, in most cases encoding secreted proteins, constituted 68% of the total mRNA in pancreas. This demonstrates the extreme specialization of the pancreas for production of secreted proteins. Among the elevated expression profiles, several previously not described proteins were identified, both in endocrine cells (CFC1, FAM159B, RBPJL and RGS9) and exocrine glandular cells (AQP12A, DPEP1, GATM and ERP27). In summary, we provide a global analysis of the pancreas transcriptome and proteome with a comprehensive list of genes and proteins with elevated expression in pancreas. This list represents an important starting point for further studies of the molecular repertoire of pancreatic cells and their relation to disease states or treatment effects.
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| 2. |
- Djureinovic, Dijana, et al.
(författare)
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The human testis-specific proteome defined by transcriptomics and antibody-based profiling
- 2014
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Ingår i: Molecular human reproduction. - : Oxford University Press (OUP). - 1360-9947 .- 1460-2407. ; 20:6, s. 476-488
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Tidskriftsartikel (refereegranskat)abstract
- The testis' function is to produce haploid germ cells necessary for reproduction. Here we have combined a genome-wide transcriptomics analysis with immunohistochemistry-based protein profiling to characterize the molecular components of the testis. Deep sequencing (RNA-Seq) of normal human testicular tissue from seven individuals was performed and compared with 26 other normal human tissue types. All 20 050 putative human genes were classified into categories based on expression patterns. The analysis shows that testis is the tissue with the most tissue-specific genes by far. More than 1000 genes show a testis-enriched expression pattern in testis when compared with all other analyzed tissues. Highly testis enriched genes were further characterized with respect to protein localization within the testis, such as spermatogonia, spermatocytes, spermatids, sperm, Sertoli cells and Leydig cells. Here we present an immunohistochemistry-based analysis, showing the localization of corresponding proteins in different cell types and various stages of spermatogenesis, for 62 genes expressed at > 50-fold higher levels in testis when compared with other tissues. A large fraction of these genes were unexpectedly expressed in early stages of spermatogenesis. In conclusion, we have applied a genome-wide analysis to identify the human testis-specific proteome using transcriptomics and antibody-based protein profiling, providing lists of genes expressed in a tissue-enriched manner in the testis. The majority of these genes and proteins were previously poorly characterised in terms of localization and function, and our list provides an important starting point to increase our molecular understanding of human reproductive biology and disease.
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| 3. |
- Edlund, Karolina, et al.
(författare)
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CD99 is a novel prognostic stromal marker in non-small cell lung cancer
- 2012
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Ingår i: International Journal of Cancer. - : Wiley. - 0020-7136 .- 1097-0215. ; 131:10, s. 2264-2273
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Tidskriftsartikel (refereegranskat)abstract
- The complex interaction between cancer cells and the microenvironment plays an essential role in all stages of tumourigenesis. Despite the significance of this interplay, alterations in protein composition underlying tumourstroma interactions are largely unknown. The aim of this study was to identify stromal proteins with clinical relevance in non-small cell lung cancer (NSCLC). A list encompassing 203 stromal candidate genes was compiled based on gene expression array data and available literature. The protein expression of these genes in human NSCLC was screened using the Human Protein Atlas. Twelve proteins were selected that showed a differential stromal staining pattern (BGN, CD99, DCN, EMILIN1, FBN1, PDGFRB, PDLIM5, POSTN, SPARC, TAGLN, TNC and VCAN). The corresponding antibodies were applied on tissue microarrays, including 190 NSCLC samples, and stromal staining was correlated with clinical parameters. Higher stromal expression of CD99 was associated with better prognosis in the univariate (p = 0.037) and multivariate (p = 0.039) analysis. The association was independent from the proportion of tumour stroma, the fraction of inflammatory cells and clinical and pathological parameters like stage, performance status and tumour histology. The prognostic impact of stromal CD99 protein expression was confirmed in an independent cohort of 240 NSCLC patients (p = 0.008). Furthermore, double-staining confocal fluorescence microscopy showed that CD99 was expressed in stromal lymphocytes as well as in cancer-associated fibroblasts. Based on a comprehensive screening strategy the membrane protein CD99 was identified as a novel stromal factor with clinical relevance. The results support the concept that stromal properties have an important impact on tumour progression.
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| 4. |
- Fagerberg, Linn, et al.
(författare)
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Analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody-based proteomics
- 2014
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Ingår i: Molecular & Cellular Proteomics. - 1535-9476 .- 1535-9484. ; 13:2, s. 397-406
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Tidskriftsartikel (refereegranskat)abstract
- Global classification of the human proteins with regards to spatial expression patterns across organs and tissues is important for studies of human biology and disease. Here, we used a quantitative transcriptomics analysis (RNA-Seq) to classify the tissue-specific expression of genes across a representative set of all major human organs and tissues and combined this analysis with antibody- based profiling of the same tissues. To present the data, we launch a new version of the Human Protein Atlas that integrates RNA and protein expression data corresponding to 80% of the human protein-coding genes with access to the primary data for both the RNA and the protein analysis on an individual gene level. We present a classification of all human protein-coding genes with regards to tissue-specificity and spatial expression pattern. The integrative human expression map can be used as a starting point to explore the molecular constituents of the human body.
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| 5. |
- Larsson, Karin, et al.
(författare)
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Novel antigen design for the generation of antibodies to G-protein-coupled receptors
- 2011
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Ingår i: JIM - Journal of Immunological Methods. - : Elsevier BV. - 0022-1759 .- 1872-7905. ; 370:1-2, s. 14-23
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Tidskriftsartikel (refereegranskat)abstract
- Antibodies are important tools for the study of G-protein-coupled receptors, key proteins in cellular signaling. Due to their large hydrophobic membrane spanning regions and often very short loops exposed on the surface of the cells, generation of antibodies able to recognize the receptors in the endogenous environment has been difficult. Here, we describe an antigen-design method where the extracellular loops and N-terminus are combined to a single antigen for generation of antibodies specific to three selected GPCRs: NPY5R, B2ARN and GLP1R. The design strategy enabled straightforward antigen production and antibody generation. Binding of the antibodies to intact receptors was analyzed using flow cytometry and immunofluorescence based confocal microscopy on A-431 cells overexpressing the respective GPCR. The antibody-antigen interactions were characterized using epitope mapping, and the antibodies were applied in immunohistochemical staining of human tissues. Most of the antibodies showed specific binding to their respective overexpressing cell line but not to the non-transfected cells, thus indicating binding to their respective target receptor. The epitope mapping showed that sub-populations within the purified antibody pool recognized different regions of the antigen. Hence, the genetic combination of several different epitopes enables efficient generation of specific antibodies with potential use in several applications for the study of endogenous receptors.
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| 6. |
- Lindskog Bergström, Cecilia, 1981-
(författare)
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Tissue Microarrays for Analysis of Expression Patterns
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Proteins are essential building blocks in every living cell, and since the complete human genome was sequenced in 2004, researchers have attempted to map the human proteome, which is the functional representation of the genome. One such initiative is the Human Protein Atlas programme (HPA), which generates monospecific antibodies towards all human proteins and uses these for high-throughput tissue profiling on tissue microarrays (TMAs). The results are publically available at the website www.proteinatlas.org.In this thesis, TMAs were used for analysis of expression patterns in various research areas. Different search queries in the HPA were tested and evaluated, and a number of potential biomarkers were identified, e.g. proteins exclusively expressed in islets of Langerhans, but not in exocrine glandular cells or other abdominal organs close to pancreas. The identified candidates were further analyzed on TMAs with pancreatic tissues from normal and diabetic individuals, and colocalization studies with insulin and glucagon revealed that several of the investigated proteins (DGCR2, GBF1, GPR44 and SerpinB10) appeared to be beta cell specific. Moreover, a set of proteins differentially expressed in lung cancer stroma was further analyzed on a clinical lung cancer cohort in the TMA format, and one protein (CD99) was significantly associated with survival. In addition, TMAs with tissue samples from different species were generated, e.g. for mapping of influenza virus attachment in various human and avian tissues. The results showed that the gull influenza virus H16N3 attached to human respiratory tract and eye, suggesting possible transmission of the virus between gull and human. TMAs were also used for analysis of protein expression differences between humans and other primates, and two proteins (TCF3 and SATB2) proved to be significantly differentially expressed on the human lineage at both the protein level and the RNA level. In conclusion, this thesis exemplifies the potential of the TMA technology, which can be used for analysis of expression patterns in a large variety of research fields, such as biomarker discovery, influenza virus research or further understanding of human evolution.
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| 7. |
- Lindskog, Cecilia, et al.
(författare)
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Analysis of Candidate Genes for Lineage-Specific Expression Changes in Humans and Primates
- 2014
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 13:8, s. 3596-3606
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Tidskriftsartikel (refereegranskat)abstract
- RUNX2, a gene involved in skeletal development, has previously been shown to be potentially affected by positive selection during recent human evolution. Here we have used antibody-based proteomics to characterize potential differences in expression patterns of RUNX2 interacting partners during primate evolution. Tissue microarrays consisting of a large set of normal tissues from human and macaque were used for protein profiling of 50 RUNX2 partners with immunohistochemistry. Eleven proteins (AR, CREBBP, EP300, FGF2, HDAC3, JUN, PRKD3, RUNX1, SATB2, TCF3, and YAP1) showed differences in expression between humans and macaques. These proteins were further profiled in tissues from chimpanzee, gorilla, and orangutan, and the corresponding genes were analyzed with regard to genomic features. Moreover, protein expression data were compared with previously obtained RNA sequencing data from six different organs. One gene (TCF3) showed significant expression differences between human and macaque at both the protein and RNA level, with higher expression in a subset of germ cells in human testis compared with macaque. In conclusion, normal tissues from macaque and human showed differences in expression of some RUNX2 partners that could be mapped to various defined cell types. The applied strategy appears advantageous to characterize the consequences of altered genes selected during evolution.
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| 8. |
- Lindskog, Cecilia, et al.
(författare)
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Antibody-based proteomics for discovery and exploration of proteins expressed in pancreatic islets
- 2010
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Ingår i: Discovery Medicine. - 1539-6509 .- 1944-7930. ; 9:49, s. 565-578
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Tidskriftsartikel (refereegranskat)abstract
- Abnormal glucose tolerance and deviant blood glucose levels are late stage clinical parameters that signify diabetes mellitus. To be able to diagnose the disease at an earlier stage and develop new tools for beta cell imaging, new molecular markers are needed. In the present study, five proteins highly expressed in pancreatic islets with no expression in the surrounding exocrine glandular cells of pancreas, and one protein with the opposite expression pattern, were identified by searches in the Human Protein Atlas (www.proteinatlas.org). The proteins were analyzed immunohistochemically on a specially designed tissue microarray, containing isolated human islets and pancreatic tissues with different characteristics, and compared to the expression of previously known markers of endocrine and exocrine pancreatic cells. Of the five novel endocrine markers, tetraspanin-7 was identified as a membrane-bound protein with exclusive positivity in islet cells. Also beta-2-microglobulin and ubiquitin carboxyl-terminal hydrolase isozyme L1 were expressed in a majority of islet cells, whereas sad1/unc-84 domain-containing protein 1 and beta-1,3-glucuronyltransferase 1 were positive in a smaller subset of islet cells. The potential exocrine marker galectin-2 was expressed in both exocrine acinary cells and pancreatic ductal cells, with no or low positivity in islet cells. In conclusion, antibody-based proteomics and specially designed tissue microarrays enable identification and exploration of novel proteins with differential expression in pancreatic islets. Here we describe 5 candidate proteins for further investigation of their physiological role and potential involvement in the pathogenesis of diabetes. One of these proteins, tetraspanin-7, is expressed on the cell membrane and could thus be a potential candidate for future development of tracers for beta cell imaging.
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| 9. |
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| 10. |
- Lindskog, Cecilia, et al.
(författare)
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Novel pancreatic beta cell-specific proteins : Antibody-based proteomics for identification of new biomarker candidates
- 2012
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Ingår i: Journal of Proteomics. - : Elsevier BV. - 1874-3919 .- 1876-7737. ; 75:9, s. 2611-2620
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Tidskriftsartikel (refereegranskat)abstract
- Beta cell-specific surface targets are required for non-invasive monitoring of beta cell mass, which could be used for evaluation of new diabetes treatments as well as to help unravel pathogenic mechanisms underlying beta cell dysfunction. By antibody-based proteomics, we have identified and explored a set of islet cell-specific proteins. A search algorithm in the Human Protein Atlas was set up for identification of islet-specific proteins that yielded 27 hits, of which twelve showed a clear membranous expression pattern or had predicted transmembrane regions. The specificity of the identified proteins was investigated by immunohistochemical staining of pancreas sections from diabetic and non-diabetic subjects. No expression of these antigens could be detected in the exocrine pancreas. Colocalization with insulin and glucagon was further determined by confocal microscopy using isolated human islets. All antibodies specifically stained human islets and colocalization analysis revealed that four proteins were exclusively expressed in beta cells. Importantly, these antibodies were negative in sections from subjects with long-standing type 1 diabetes. In the present study, we present four proteins; DGCR2, GBF1, GPR44 and SerpinB10, the expression of which has not previously been described in beta cells.
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