| 1. |
- Malm, Johan, et al.
(författare)
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Developments in biobanking workflow standardization providing sample integrity and stability
- 2013
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Ingår i: Journal of Proteomics. - : Elsevier BV. - 1874-3919 .- 1876-7737. ; 95:SI, s. 38-45
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Tidskriftsartikel (refereegranskat)abstract
- Recommendations and outlines for standardization in biobanking processes are presented by a research team with long-term experience in clinical studies. These processes have important bearing on the use of samples in developing assays. These measurements are useful to document states of health and disease that are beneficial for academic research, commercial healthcare, drug development industry and government regulating agencies. There is a need for increasing awareness within proteomic and genomic communities regarding the basic concepts of collecting, storing and utilizing clinical samples. Quality control and sample suitability for analysis need to be documented and validated to ensure data integrity and establish contexts for interpretation of results. Standardized methods in proteomics and genomics are required to be practiced throughout the community allowing datasets to be comparable and shared for analysis. For example, sample processing of thousands of clinical samples, performed in 384 high-density sample tube systems in a fully automated workflow, preserves sample content and is presented showing validation criteria. Large studies will be accompanied by biological and molecular information with corresponding clinical records from patients and healthy donors. These developments position biobanks of human patient samples as an increasingly recognized major asset in disease research, future drug development and within patient care. Biological significance: The current manuscript is of major relevance to the proteomic and genomic fields, as it outlines the standardization aspects of biobanking and the requirements that are needed to run future clinical studies that will benefit the patients where OMICS science will play a major role. A global view of the field is given where best practice and conventional acceptances are presented along with ongoing large-scale biobanking projects. The authors represent broadly stakeholders that cover the academic, pharma, biotech and healthcare fields with extensive experience and deliveries. This contribution will be a milestone paper to the proteomic and genomic scientists to present data in the future that will have impact to the life science area.This article is part of a Special Issue entitled: Standardization and Quality Control in Proteomics.
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| 2. |
- Carlsohn, Elisabet, et al.
(författare)
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Characterization of the outer membrane protein profile from disease-related Helicobacter pylori isolates by subcellular fractionation and nano-LC FT-ICR MS analysis
- 2006
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Ingår i: J Proteome Res. - : American Chemical Society (ACS). - 1535-3893. ; 5:11, s. 3197-204
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Because of the important role of membrane proteins in adhesion, invasion, and intracellular survival of pathogens in the host, membrane proteins are of potential interest in the search for drug targets or biomarkers. We have established a mass spectrometry-based method that allows characterization of the outer membrane protein (OMP) profile of clinical isolates from of the human gastric pathogen Helicobacter pylori. Subcellular fractionation and one-dimensional gel electrophoresis (1D-GE) analysis was combined with nano-liquid chromatography Fourier transform-ion cyclotron resonance mass spectrometry (nano-LC FT-ICR MS) and tandem mass spectrometry (MS/MS) analysis of fifteen H. pylori strains associated either with duodenal ulcers, gastric cancer, or isolated from asymptomatic H. pylori infected carriers. Over 60 unique membrane or membrane-associated proteins, including 30 of the 33 theoretically predicted OMPs, were identified from the strains. Several membrane proteins, including Omp11 and BabA, were found to be expressed by all strains. In the search for clinical markers we found that Omp26 was expressed by all disease-related strains but was only present in one out of five strains from asymptomatic carriers, which makes Omp26 a potential target for further investigation in the search for proteins unique to disease-related H. pylori strains. In addition, presence of Omp30 and absence of Omp6 seemed to be associated with H. pylori strains causing duodenal ulcer.
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| 3. |
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| 4. |
- Carlsohn, Elisabet, et al.
(författare)
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HpaA is essential for Helicobacter pylori colonization in mice
- 2006
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Ingår i: Infect Immun. ; 74:2, s. 920-6
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Tidskriftsartikel (refereegranskat)abstract
- Infection with the human gastric pathogen Helicobacter pylori can give rise to chronic gastritis, peptic ulcer, and gastric cancer. All H. pylori strains express the surface-localized protein HpaA, a promising candidate for a vaccine against H. pylori infection. To study the physiological importance of HpaA, a mutation of the hpaA gene was introduced into a mouse-adapted H. pylori strain. To justify that the interruption of the hpaA gene did not cause any polar effects of downstream genes or was associated with a second site mutation, the protein expression patterns of the mutant and wild-type strains were characterized by two different proteomic approaches. Two-dimensional differential in-gel electrophoresis analysis of whole-cell extracts and subcellular fractionation combined with nano-liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometry for outer membrane protein profiling revealed only minor differences in the protein profile between the mutant and the wild-type strains. Therefore, the mutant strain was tested for its colonizing ability in a well-established mouse model. While inoculation with the wild-type strain resulted in heavily H. pylori-infected mice, the HpaA mutant strain was not able to establish colonization. Thus, by combining proteomic analysis and in vivo studies, we conclude that HpaA is essential for the colonization of H. pylori in mice.
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| 5. |
- Carlsohn, Elisabet
(författare)
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Mass spectrometry-based proteomic strategies applied to Helicobacter pylori. A Search for candidate vaccine antigens
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis describes the development and refinement of proteomic strategies and the application of these on the human gastric pathogen H. pylori. The purpose was to identify proteins, and in particular membrane proteins, as potential targets for vaccine development. A semi-preparative method based on liquid-phase separation of whole cell extracts was found to be useful. However, in order to perform a more comprehensive analysis of the outer membrane protein profile, and to identify proteins specific for disease-related H. pylori strains, a new strategy combining subcellular fractionation with high-sensitivity nano-liquid chromatography Fourier transform-ion cyclotron resonance mass spectrometry (nano-LC FT-ICR MS) analysis was used. Differences among outer membrane proteins (OMPs) were compared in clinical bacterial isolates of asymptomatic carriers and of patients with duodenal ulcer or gastric cancer. Several potential targets for a vaccine were identified and proteins that may be used as diagnostic markers or should be subjected to more detailed studies were identified. These results document the importance of using nano-LC coupled to the powerful FT-ICR MS method for high confidence identification of low-abundance components of a complex mixture. The protein expression patterns of the mouse-adapted H. pylori strain SS1 and its isogenic HpaA mutant strain were examined using the two strategies. Whole cell extracts were analyzed by traditional 2D- gel electrophoresis (2D-GE) and differential in-gel electrophoresis (DIGE) technology, and the OMP profiles were compared using the fractionation and nano-LC MS approach. Only minor differences in the protein expression pattern were found between the two strains. HpaA was identified in the SS1 strain, but could not be detected in the mutant strain, although the low-abundance OMP BabA was readily identified. Infection studies in mice demonstrated that HpaA is essential for H. pylori colonization, and this protein is therefore a candidate vaccine antigen. In order to evaluate the potential of using mass spectrometry to localize surface-exposed peptides of a protein (potential immunogenic epitopes), we performed chemical cross-linking studies of the urease complex of H. pylori. This protein is easy to prepare and the known three-dimensional structure facilitates the interpretation of results by molecular modeling. Although cross-linked peptides were identified, the conclusion is that this potentially helpful technique requires additional developmental efforts to be practically useful.
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| 6. |
- Karlsson, Roger, 1975, et al.
(författare)
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Strain-level typing and identification of bacteria using mass spectrometry-based proteomics.
- 2012
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Ingår i: Journal of proteome research. - : American Chemical Society (ACS). - 1535-3907 .- 1535-3893. ; 11:5, s. 2710-20
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Tidskriftsartikel (refereegranskat)abstract
- Because of the alarming expansion in the diversity and occurrence of bacteria displaying virulence and resistance to antimicrobial agents, it is increasingly important to be able to detect these microorganisms and to differentiate and identify closely related species, as well as different strains of a given species. In this study, a mass spectrometry proteomics approach is applied, exploiting lipid-based protein immobilization (LPI), wherein intact bacterial cells are bound, via membrane-gold interactions, within a FlowCell. The bound cells are subjected to enzymatic digestion for the generation of peptides, which are subsequently identified, using LC-MS. Following database matching, strain-specific peptides are used for subspecies-level discrimination. The method is shown to enable a reliable typing and identification of closely related strains of the same bacterial species, herein illustrated for Helicobacter pylori .
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| 7. |
- Kristjansdottir, Björg, et al.
(författare)
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Potential tumor biomarkers identified in ovarian cyst fluid by quantitative proteomic analysis, iTRAQ.
- 2013
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Ingår i: Clinical proteomics. - : Springer Science and Business Media LLC. - 1542-6416 .- 1559-0275. ; 10:1
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Tidskriftsartikel (refereegranskat)abstract
- Epithelial-derived ovarian adenocarcinoma (EOC) is the most deadly gynecologic tumor, and the principle cause of the poor survival rate is diagnosis at a late stage. Screening and diagnostic biomarkers with acceptable specificity and sensitivity are lacking. Ovarian cyst fluid should harbor early ovarian cancer biomarkers because of its closeness to the tumor. We investigated ovarian cyst fluid as a source for discovering biomarkers for use in the diagnosis of EOC.
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| 8. |
- Mahdavi, J., et al.
(författare)
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A novel O-linked glycan modulates Campylobacter jejuni major outer membrane protein-mediated adhesion to human histo-blood group antigens and chicken colonization
- 2014
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Ingår i: Open Biology. - : The Royal Society. - 2046-2441. ; 4:1
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Tidskriftsartikel (refereegranskat)abstract
- Campylobacter jejuni is an important cause of human foodborne gastroenteritis; strategies to prevent infection are hampered by a poor understanding of the complex interactions between host and pathogen. Previous work showed that C. jejuni could bind human histo-blood group antigens (BgAgs) in vitro and that BgAgs could inhibit the binding of C. jejuni to human intestinal mucosa ex vivo. Here, the major flagella subunit protein (FlaA) and the major outer membrane protein (MOMP) were identified as BgAg-binding adhesins in C. jejuni NCTC11168. Significantly, the MOMP was shown to be O-glycosylated at Thr(268); previously only flagellin proteins were known to be O-glycosylated in C. jejuni. Substitution of MOMP Thr(268) led to significantly reduced binding to BgAgs. The O-glycan moiety was characterized as Gal(beta 1-3)-GalNAc(beta 1-4)-GalNAc(beta 1-4)-GalNAca1-Thr(268); modelling suggested that O-glycosylation has a notable effect on the conformation of MOMP and this modulates BgAg-binding capacity. Glycosylation of MOMP at Thr(268) promoted cell-to-cell binding, biofilm formation and adhesion to Caco-2 cells, and was required for the optimal colonization of chickens by C. jejuni, confirming the significance of this O-glycosylation in pathogenesis.
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| 9. |
- Nilsson, C. L., et al.
(författare)
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Chromosome 19 Annotations with Disease Speciation: A First Report from the Global Research Consortium
- 2013
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 12:1, s. 134-149
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Tidskriftsartikel (refereegranskat)abstract
- A first research development progress report of the Chromosome 19 Consortium with members from Sweden, Norway, Spain, United States, China and India, a part of the Chromosome-centric Human Proteome Project (C-HPP) global initiative, is presented (http://www.c-hpp.org). From the chromosome 19 peptide-targeted library constituting 6159 peptides, a pilot study was conducted using a subset with 125 isotope-labeled peptides. We applied an annotation strategy with triple quadrupole, ESI-Qtrap, and MALDI mass spectrometry platforms, comparing the quality of data within and in between these instrumental set-ups. LC–MS conditions were outlined by multiplex assay developments, followed by MRM assay developments. SRM was applied to biobank samples, quantifying kallikrein 3 (prostate specific antigen) in plasma from prostate cancer patients. The antibody production has been initiated for more than 1200 genes from the entire chromosome 19, and the progress developments are presented. We developed a dedicated transcript microarray to serve as the mRNA identifier by screening cancer cell lines. NAPPA protein arrays were built to align with the transcript data with the Chromosome 19 NAPPA chip, dedicated to 90 proteins, as the first development delivery. We have introduced an IT-infrastructure utilizing a LIMS system that serves as the key interface for the research teams to share and explore data generated within the project. The cross-site data repository will form the basis for sample processing, including biological samples as well as patient samples from national Biobanks.
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| 10. |
- Nilsson, C. L., et al.
(författare)
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Use of ENCODE Resources to Characterize Novel Proteoforms and Missing Proteins in the Human Proteome
- 2015
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 14:2, s. 603-608
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Tidskriftsartikel (refereegranskat)abstract
- We describe the utility of integrated strategies that employ both translation of ENCODE data and major proteomic technology pillars to improve the identification of the "missing proteins", novel proteoforms, and PTMs. On one hand, databases in combination with bioinformatic tools are efficiently utilized to establish microarray-based transcript analysis and supply rapid protein identifications in clinical samples. On the other hand, sequence libraries are the foundation of targeted protein identification and quantification using mass spectrometric and immunoaffinity techniques. The results from combining proteoENCODEdb searches with experimental mass spectral data indicate that some alternative splicing forms detected at the transcript level are in fact translated to proteins. Our results provide a step toward the directives of the C-HPP initiative and related biomedical research.
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