1.
Stella, Roberto, et al.
(författare)
Relative Quantification of Membrane Proteins in Wild-Type and Prion Protein (PrP)-Knockout Cerebellar Granule Neurons
2012
Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 11:2, s. 523-536
Tidskriftsartikel (refereegranskat) abstract
Approximately 25% of eukaryotic proteins possessing homology to at least two trans membrane domains are predicted to be embedded in biological membranes. Nevertheless, this group of proteins is not usually well represented in proteome-wide experiments due to their refractory nature. Here we present a quantitative mass spectrometry-based comparison of membrane protein expression in cerebellar granule neurons grown in primary culture that were isolated from wild-type mice and mice lacking the cellular prion protein. This protein is a cell-surface glycoprotein that is mainly expressed in the central nervous system and is involved in several neurodegenerative disorders, though its physiological role is unclear. We used a low specificity enzyme a-chymotrypsin to digest membrane proteins preparations that had been separated by SDS-PAGE. The resulting peptides were labeled with tandem mass tags and analyzed by MS. The differentially expressed proteins identified using this approach were further analyzed by multiple reaction monitoring to confirm the expression level changes.
2.
Deutsch, Eric W., et al.
(författare)
TraML: a standard format for exchange of selected reaction monitoring transition lists
2012
Ingår i: Molecular & Cellular Proteomics. - 1535-9484. ; 11:4, s. 111-015040
Tidskriftsartikel (refereegranskat) abstract
Abstract in UndeterminedTargeted proteomics via selected reaction monitoring (SRM) is a powerful mass spectrometric technique affording higher dynamic range, increased specificity and lower limits of detection than other shotgun mass spectrometry methods when applied to proteome analyses. However, it involves selective measurement of predetermined analytes, which requires more preparation in the form of selecting appropriate signatures for the proteins and peptides that are to be targeted. There is a growing number of software programs and resources for selecting optimal transitions and the instrument settings used for the detection and quantification of the targeted peptides, but the exchange of this information is hindered by a lack of a standard format. We have developed a new standardized format, called TraML, for encoding transition lists and associated metadata. In addition to introducing the TraML format, we demonstrate several implementations across the community, and provide semantic validators, extensive documentation, and multiple example instances to demonstrate correctly written documents. Widespread use of TraML will facilitate the exchange of transitions, reduce time spent handling incompatible list formats, increase the reusability of previously optimized transitions, and thus accelerate the widespread adoption of targeted proteomics via SRM.
3.
Martens, Lennart, et al.
(författare)
mzML - a community standard for mass Spectrometry data
2011
Ingår i: Molecular & Cellular Proteomics. - 1535-9484. ; 10:1, s. 1-000133
Tidskriftsartikel (refereegranskat) abstract
Mass spectrometry is a fundamental tool for discovery and analysis in the life sciences. With the rapid advances in mass spectrometry technology and methods, it has become imperative to provide a standard output format for mass spectrometry data that will facilitate data sharing and analysis. Initially, the efforts to develop a standard format for mass spectrometry data resulted in multiple formats, each designed with a different underlying philosophy. To resolve the issues associated with having multiple formats, vendors, researchers, and software developers convened under the banner of the HUPO PSI to develop a single standard. The new data format incorporated many of the desirable technical attributes from the previous data formats, while adding a number of improvements, including features such as a controlled vocabulary with validation tools to ensure consistent usage of the format, improved support for selected reaction monitoring data, and immediately available implementations to facilitate rapid adoption by the community. The resulting standard data format, mzML, is a well tested open-source format for mass spectrometer output files that can be readily utilized by the community and easily adapted for incremental advances in mass spectrometry technology.
4.
Kirik, Ufuk, et al.
(författare)
Antibody heavy chain variable domains of different germline gene origins diversify through different paths
2017
Ingår i: Frontiers in Immunology. - : Frontiers Media SA. - 1664-3224. ; 8:NOV
Tidskriftsartikel (refereegranskat) abstract
B cells produce antibodies, key effector molecules in health and disease. They mature their properties, including their affinity for antigen, through hypermutation events; processes that involve, e.g., base substitution, codon insertion and deletion, often in association with an isotype switch. Investigations of antibody evolution define modes whereby particular antibody responses are able to form, and such studies provide insight important for instance for development of efficient vaccines. Antibody evolution is also used in vitro for the design of antibodies with improved properties. To better understand the basic concepts of antibody evolution, we analyzed the mutational paths, both in terms of amino acid substitution and insertions and deletions, taken by antibodies of the IgG isotype. The analysis focused on the evolution of the heavy chain variable domain of sets of antibodies, each with an origin in 1 of 11 different germline genes representing six human heavy chain germline gene subgroups. Investigated genes were isolated from cells of human bone marrow, a major site of antibody production, and characterized by next-generation sequencing and an in-house bioinformatics pipeline. Apart from substitutions within the complementarity determining regions, multiple framework residues including those in protein cores were targets of extensive diversification. Diversity, both in terms of substitutions, and insertions and deletions, in antibodies is focused to different positions in the sequence in a germline gene-unique manner. Altogether, our findings create a framework for understanding patterns of evolution of antibodies from defined germline genes.
5.
Persson, Helena, et al.
(författare)
In Vitro Evolution of Antibodies Inspired by In Vivo Evolution
2018
Ingår i: Frontiers in Immunology. - : Frontiers Media S.A.. - 1664-3224. ; 9
Tidskriftsartikel (refereegranskat) abstract
In vitro generation of antibodies often requires variable domain sequence evolution to adapt the protein in terms of affinity, specificity, or developability. Such antibodies, including those that are of interest for clinical development, may have their origins in a diversity of immunoglobulin germline genes. Others and we have previously shown that antibodies of different origins tend to evolve along different, preferred trajectories. Apart from substitutions within the complementary determining regions, evolution may also, in a germline gene-origin-defined manner, be focused to residues in the framework regions, and even to residues within the protein core, in many instances at a substantial distance from the antibody's antigen-binding site. Examples of such germline origin-defined patterns of evolution are described. We propose that germline gene-preferred substitution patterns offer attractive alternatives that should be considered in efforts to evolve antibodies intended for therapeutic use with respect to appropriate affinity, specificity, and product developability. We also hypothesize that such germline gene-origin-defined in vitro evolution hold potential to result in products with limited immunogenicity, as similarly evolved antibodies will be parts of conventional, in vivo-generated antibody responses and thus are likely to have been seen by the immune system in the past.
6.
Hosseini, Sara, et al.
(författare)
Comparative proteomic analysis of hyphae and germinating cysts of Phytophthora pisi and Phytophthora sojae.
2015
Ingår i: Journal of Proteomics. - : Elsevier BV. - 1874-3919 .- 1876-7737. ; 117, s. 24-40
Tidskriftsartikel (refereegranskat) abstract
The recently described oomycete pathogen Phytophthora pisi causes root rot on pea and faba bean, while the closely related Phytophthora sojae is the causal agent of soybean root and stem rot. Differences in the pathogenicity factor repertoires that enable the two species to have distinct host specificity towards pea and soybean, were studied using tandem mass spectrometry in a global proteome study of hyphae and germinating cysts in P. pisi and P. sojae. In total 2775 proteins from P. pisi and 2891 proteins from P. sojae were identified. Fifty-eight orthologous proteins were more abundant in germinated cysts of both pathogens and thus identified as candidate proteins for the infective stage. Several of these proteins were associated with lipid transport and metabolism, and energy production. Twenty-three orthologous proteins were more abundant in hyphae of both pathogens and thus identified as candidate proteins for vegetative growth. Proteins uniquely present in germinating cysts of either P. pisi or P. sojae were considered as candidates for species-specific pathogenicity factors that may be involved in host specificity. Among these proteins were serine proteases, membrane transporters and a berberine-like protein. These results significantly expand the knowledge of the expressed proteome in P. pisi and P. sojae.
7.
8.
Karhumaa, Kaisa, et al.
(författare)
Proteome analysis of the xylose-fermenting mutant yeast strain TMB 3400.
2009
Ingår i: Yeast. - : Wiley. - 1097-0061 .- 0749-503X. ; 26:7, s. 371-382
Tidskriftsartikel (refereegranskat) abstract
Xylose fermentation in yeast has been a target of research for years, yet not all the factors that may affect xylose fermentation perfomance of yeast strains are known. In this study, the mutant S. cerevisiae strain TMB 3400, which has good xylose fermentation properties, was compared with its parental strain to examine the factors behind the improved xylose utilization at protein level. The proteome of the parental and the mutant strains were characterized by difference in gel electrophoresis (DiGE) to quantitatively identify proteins that are expressed at altered levels in the mutant. The most significant changes detected by proteome analysis were the 6-10-fold increased levels of xylose reductase, xylitol dehydrogenase and transketolase (Tkl1) in the mutant, which is in accordance with previous knowledge about xylose metabolism in yeast. The level of acetaldehyde dehydrogenase (Ald6) was also significantly increased. In addition, several proteins homologous to proteins from yeast species other than S. cerevisiae were identified in both strains, demonstrating the genetic heterogeneity of industrial yeast strains. The results were also compared with a previously reported transcription analysis performed with identical experimental set-up; however, very little correlation between the two datasets was observed. The results of the proteome analysis were in good agreement with a parallel study in which rationally designed overexpression of XR, XDH and the non-oxidative pentose phosphate pathway resulted in similar improvement in xylose utilization, which demonstrates the usefulness of proteome analysis for the identification of target genes for further metabolic engineering strategies in industrial yeast strains. Copyright (c) 2009 John Wiley & Sons, Ltd.
9.
Burra, Dharani, et al.
(författare)
Phosphite-induced changes of the transcriptome and secretome in Solanum tuberosum leading to resistance against Phytophthora infestans
2014
Ingår i: BMC Plant Biology. - : Springer Science and Business Media LLC. - 1471-2229. ; 14
Tidskriftsartikel (refereegranskat) abstract
Background: Potato late blight caused by the oomycete pathogen Phytophthora infestans can lead to immense yield loss. We investigated the transcriptome of Solanum tubersoum (cv. Desiree) and characterized the secretome by quantitative proteomics after foliar application of the protective agent phosphite. We also studied the distribution of phosphite in planta after application and tested transgenic potato lines with impaired in salicylic and jasmonic acid signaling. Results: Phosphite had a rapid and transient effect on the transcriptome, with a clear response 3 h after treatment. Strikingly this effect lasted less than 24 h, whereas protection was observed throughout all time points tested. In contrast, 67 secretome proteins predominantly associated with cell-wall processes and defense changed in abundance at 48 h after treatment. Transcripts associated with defense, wounding, and oxidative stress constituted the core of the phosphite response. We also observed changes in primary metabolism and cell wall-related processes. These changes were shown not to be due to phosphate depletion or acidification caused by phosphite treatment. Of the phosphite-regulated transcripts 40% also changed with beta-aminobutyric acid (BABA) as an elicitor, while the defence gene PR1 was only up-regulated by BABA. Although phosphite was shown to be distributed in planta to parts not directly exposed to phosphite, no protection in leaves without direct foliar application was observed. Furthermore, the analysis of transgenic potato lines indicated that the phosphite-mediated resistance was independent of the plant hormones salicylic and jasmonic acid. Conclusions: Our study suggests that a rapid phosphite-triggered response is important to confer long-lasting resistance against P. infestans and gives molecular understanding of its successful field applications.
10.
Chawade, Aakash, et al.
(författare)
Normalyzer: A Tool for Rapid Evaluation of Normalization Methods for Omics Data Sets
2014
Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 13:6, s. 3114-3120
Tidskriftsartikel (refereegranskat) abstract
High-throughput omics data often contain systematic biases introduced during various steps of sample processing and data generation. As the source of these biases is usually unknown, it is difficult to select an optimal normalization method for a given data set. To facilitate this process, we introduce the open-source tool "Normalyzer". It normalizes the data with 12 different normalization methods and generates a report with several quantitative and qualitative plots for comparative evaluation of different methods. The usefulness of Normalyzer is demonstrated with three different case studies from quantitative proteomics and transcriptomics. The results from these case studies show that the choice of normalization method strongly influences the outcome of downstream quantitative comparisons. Normalyzer is an R package and can be used locally or through the online implementation at http://quantitativeproteomics.org/normalyzer.
