| 1. |
- Danielsson, Frida, et al.
(författare)
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RNA Deep Sequencing as a Tool for Selection of Cell Lines for Systematic Subcellular Localization of All Human Proteins
- 2013
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 12:1, s. 231-239
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Tidskriftsartikel (refereegranskat)abstract
- One of the major challenges of a chromosome-centric proteome project is to explore in a systematic manner the potential proteins identified from the chromosomal genome sequence, but not yet characterized on a protein level. Here, we describe the use of RNA deep sequencing to screen human cell lines for RNA profiles and to use this information to select cell lines suitable for characterization of the corresponding gene product. In this manner, the subcellular localization of proteins can be analyzed systematically using antibody-based confocal microscopy. We demonstrate the usefulness of selecting cell lines with high expression levels of RNA transcripts to increase the likelihood of high quality immunofluorescence staining and subsequent successful subcellular localization of the corresponding protein. The results show a path to combine transcriptomics with affinity proteomics to characterize the proteins in a gene- or chromosome-centric manner.
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| 2. |
- Hjelm, Barbara, et al.
(författare)
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Generation of monospecific antibodies based on affinity capture of polyclonal antibodies
- 2011
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 20:11, s. 1824-1835
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Tidskriftsartikel (refereegranskat)abstract
- A method is described to generate and validate antibodies based on mapping the linear epitopes of a polyclonal antibody followed by sequential epitope-specific capture using synthetic peptides. Polyclonal antibodies directed towards four proteins RBM3, SATB2, ANLN, and CNDP1, potentially involved in human cancers, were selected and antibodies to several non-overlapping epitopes were generated and subsequently validated by Western blot, immunohistochemistry, and immunofluorescence. For all four proteins, a dramatic difference in functionality could be observed for these monospecific antibodies directed to the different epitopes. In each case, at least one antibody was obtained with full functionality across all applications, while other epitope-specific fractions showed no or little functionality. These results present a path forward to use the mapped binding sites of polyclonal antibodies to generate epitope-specific antibodies, providing an attractive approach for large-scale efforts to characterize the human proteome by antibodies.
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| 3. |
- Kalderén, Christina, et al.
(författare)
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CCL2 mediates anti-fibrotic effects in human fibroblasts independently of CCR2
- 2014
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Ingår i: International Immunopharmacology. - : Elsevier BV. - 1567-5769 .- 1878-1705. ; 20:1, s. 66-73
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Tidskriftsartikel (refereegranskat)abstract
- CCL2 is known for its major role as a chemoattractant of monocytes for immunological surveillance and to site of inflammation. CCL2 acts mainly through the G-protein-coupled receptor CCR2 but has also been described to mediate its effects independently of this receptor in vitro and in vivo. Emerging pieces of evidence indicate that the CCL2/CCR2 axis is involved in fibrotic diseases, such as increased plasma levels of CCL2 and the presence of CCL2-hyperresponsive fibroblasts explanted from patients with systemic sclerosis and idiopathic pulmonary fibrosis. One of the profibrotic key mediators is the myofibroblast characterized by overexpression of alpha-smooth muscle actin and collagen I. However, the correlation between the CCL2/CCR2 axis and the activation of fibroblasts is not yet fully understood. We have screened human fibroblasts of various origins, human pulmonary fibroblasts (HPF), human fetal lung fibroblasts (HFL-1) and primary preadipocytes (SPF-1) in regard to CCL2 stimulated fibrotic responses. Surprisingly we found that CCL2 mediates anti-fibrotic effects independently of CCR2 in human fibroblasts of different origins.
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| 4. |
- Stadler, Charlotte, et al.
(författare)
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A single fixation protocol for proteome-wide immunofluorescence localization studies
- 2010
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Ingår i: Journal of Proteomics. - : Elsevier BV. - 1874-3919 .- 1876-7737. ; 73:6, s. 1067-1078
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Tidskriftsartikel (refereegranskat)abstract
- Immunofluorescence microscopy is a valuable tool for analyzing protein expression and localization at a subcellular level thus providing information regarding protein function, interaction partners and its role in cellular processes. When performing sample fixation, parameters such as difference in accessibility of proteins present in various cellular compartments as well as the chemical composition of the protein to be studied, needs to be taken into account. However, in systematic and proteome-wide efforts, a need exists for standard fixation protocol(s) that works well for the majority of all proteins independent of subcellular localization. Here, we report on a study with the goal to find a standardized protocol based on the analysis of 18 human proteins localized in 11 different organelles and subcellular structures. Six fixation protocols were tested based on either dehydration by alcohols (methanol, ethanol or iso-propanol) or cross-linking by paraformaldehyde followed by detergent permeabilization (Triton X-100 or saponin) in three human cell lines. Our results show that cross-linking is essential for proteome-wide localization studies and that cross-linking using paraformaldehyde followed by Triton X-100 permeabilization successfully can be used as a single fixation protocol for systematic studies.
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| 5. |
- Stadler, Charlotte, et al.
(författare)
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Immunofluorescence and fluorescent-protein tagging show high correlation for protein localization in mammalian cells
- 2013
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Ingår i: Nature Methods. - 1548-7091 .- 1548-7105. ; 10:4, s. 315-323
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Tidskriftsartikel (refereegranskat)abstract
- Imaging techniques such as immunofluorescence (IF) and the expression of fluorescent protein (FP) fusions are widely used to investigate the subcellular distribution of proteins. Here we report a systematic analysis of >500 human proteins comparing the localizations obtained in live versus fixed cells using FPs and IF, respectively. We identify systematic discrepancies between IF and FPs as well as between FP tagging at the N and C termini. The analysis shows that for 80% of the proteins, IF and FPs yield the same subcellular distribution, and the locations of 250 previously unlocalized proteins were determined by the overlap between the two methods. Approximately 60% of proteins localize to multiple organelles for both methods, indicating a complex subcellular protein organization. These results show that both IF and FP tagging are reliable techniques and demonstrate the usefulness of an integrative approach for a complete investigation of the subcellular human proteome.
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| 6. |
- Stadler, Charlotte, et al.
(författare)
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RNA- and Antibody-Based Profiling of the Human Proteome with Focus on Chromosome 19
- 2014
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 13:4, s. 2019-2027
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Tidskriftsartikel (refereegranskat)abstract
- An important part of the Human Proteome Project is to characterize the protein complement of the genome with antibody-based profiling. Within the framework of this effort, a new version 12 of the Human Protein Atlas (www.proteinatlas.org) has been launched, including transcriptomics data for 27 tissues and 44 cell lines to complement the protein expression data from antibody-based profiling. Besides the extensive addition of transcriptomics data, the Human Protein Atlas now contains antibody-based protein profiles for 82% of the 20 329 putative protein-coding genes. The comprehensive data resulting from RNA-seq analysis and antibody-based profiling performed within the Human Protein Atlas as well as information from UniProt were used to generate evidence summary scores for each of the 20 329 genes, of which 94% now have experimental evidence at least at transcript level. The evidence scores for all individual genes are displayed with regards to both RNA- and antibody-based protein profiles, including chromosome-centric visualizations. An analysis of the human chromosome 19 shows that similar to 43% of the genes are expressed at the transcript level in all 27 tissues analyzed, suggesting a "house-keeping" function, while 12% of the genes show a more tissue-specific pattern with enriched expression in one of the analyzed tissues only.
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| 7. |
- Stadler, Charlotte, et al.
(författare)
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Systematic validation of antibody binding and protein subcellular localization using siRNA and confocal microscopy
- 2012
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Ingår i: Journal of Proteomics. - : Elsevier BV. - 1874-3919 .- 1876-7737. ; 75:7, s. 2236-2251
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Tidskriftsartikel (refereegranskat)abstract
- We have developed a platform for validation of antibody binding and protein subcellular localization data obtained from immunofluorescence using siRNA technology combined with automated confocal microscopy and image analysis. By combining the siRNA technology with automated sample preparation, automated imaging and quantitative image analysis, a high-throughput assay has been set-up to enable confirmation of accurate protein binding and localization in a systematic manner. Here, we describe the analysis and validation of the subcellular location of 65 human proteins, targeted by 75 antibodies and silenced by 130 siRNAs. A large fraction of (80%) the subcellular locations, including locations of several previously uncharacterized proteins, could be confirmed by the significant down-regulation of the antibody signal after the siRNA silencing. A quantitative analysis was set-up using automated image analysis to facilitate studies of targets found in more than one compartment. The results obtained using the platform demonstrate that siRNA silencing in combination with quantitative image analysis of antibody signals in different compartments of the cells is an attractive approach for ensuring accurate protein localization as well as antibody binding using immunofluorescence. With a large fraction of the human proteome still unexplored, we suggest this approach to be of great importance under the continued work of mapping the human proteome on a subcellular level.
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| 8. |
- Stadler, Charlotte, 1982-
(författare)
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Towards subcellular localization of the human proteome using bioimaging
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Since the publication of the complete sequence of the human genome in 2003 there has been great interest in exploring the functions of the proteins encoded by the genes. To reveal the function of each and every protein, investigation of protein localization at the subcellular level has become a central focus in this research area, since the localization and function of a protein is closely related. The objective of the studies presented in this doctoral thesis was to systematically explore the human proteome at the subcellular level using bioimaging and to develop techniques for validation of the results obtained.A common imaging technique for protein detection is immunofluorescence (IF), where antibodies are used to target proteins in fixated cells. A fixation protocol suitable for large-scale IF studies was developed and optimized to work for a broad set of proteins. As the technique relies on antibodies, validation of their specificity to the target protein is crucial. A platform based on siRNA gene silencing in combination with IF was set-up to evaluate antibody specificity by quantitative image analysis before and after suppression of its target protein. As a proof of concept, the platform was then used for validation of 75 antibodies, proving it to be applicable for validation of antibodies in a systematic manner.Because of the fixation, there is a common concern about how well IF data reflects the in vivo subcellular distribution of proteins. To address this, 500 proteins were tagged with green fluorescent protein (GFP) and used to compare protein localization results between IF to those achieved using GFP tagged proteins in live cells. It was concluded that protein localization data from fixated cells satisfactory represented the situation in vivo and together exhibit a powerful approach for confirming localizations of yet uncharacterized proteins.Finally, a global analysis based on IF data of approximately 20 % of the human proteome was performed, providing a first overview of the subcellular landscape in three different cell lines. It was found that the intracellular distribution of proteins is complex, with many proteins occurring in several organelles. The results also confirmed the close relationship between protein function and localization, which in a way further strengthens the accuracy of the IF approach for detection of proteins at the subcellular level.
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| 9. |
- Vandamme, Peter, et al.
(författare)
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Classification of Achromobacter genogroups 2, 5, 7 and 14 as Achromobacter insuavis sp. nov., Achromobacter aegrifaciens sp. nov., Achromobacter anxifer sp. nov. and Achromobacter dolens sp. nov., respectively.
- 2013
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Ingår i: Systematic and applied microbiology. - : Elsevier BV. - 1618-0984 .- 0723-2020. ; 36:7, s. 474-482
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Tidskriftsartikel (refereegranskat)abstract
- The phenotypic and genotypic characteristics of seventeen Achromobacter strains representing MLST genogroups 2, 5, 7 and 14 were examined. Although genogroup 2 and 14 strains shared a DNA-DNA hybridization level of about 70%, the type strains of both genogroups differed in numerous biochemical characteristics and all genogroup 2 and 14 strains could by distinguished by nitrite reduction, denitrification and growth on acetamide. Given the MLST sequence divergence which identified genogroups 2 and 14 as clearly distinct populations, the availability of nrdA sequence analysis as a single locus identification tool for all Achromobacter species and genogroups, and the differential phenotypic characteristics, we propose to formally classify Achromobacter genogroups 2, 5, 7 and 14 as four novel Achromobacter species for which we propose the names Achromobacter insuavis sp. nov. (with strain LMG 26845(T) [=CCUG 62426(T)] as the type strain), Achromobacter aegrifaciens sp. nov. (with strain LMG 26852(T) [=CCUG 62438(T)] as the type strain), Achromobacter anxifer sp. nov. (with strain LMG 26857(T) [=CCUG 62444(T)] as the type strain), and Achromobacter dolens sp. nov. (with strain LMG 26840(T) [=CCUG 62421(T)] as the type strain).
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| 10. |
- Wiking, Mikaela, et al.
(författare)
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The Subcellular Protein Atlas
- 2014
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Ingår i: Molecular Biology of the Cell. - 1059-1524 .- 1939-4586. ; 25
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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