251 research outputs found
Introduction to opportunities and pitfalls in functional mass spectrometry based proteomics
With the advent of mass spectrometry based proteomics, the identification of thousands of proteins has become commonplace in biology nowadays. Increasingly, efforts have also been invested toward the detection and localization of posttranslational modifications. It is furthermore common practice to quantify the identified entities, a task supported by a panel of different methods. Finally, the results can also be enriched with functional knowledge gained on the proteins, detecting for instance differentially expressed gene ontology terms or biological pathways. In this study, we review the resources, methods and tools available for the researcher to achieve such a quantitative functional analysis. These include statistics for the post-processing of identification and quantification results, online resources and public repositories. With a focus on free but user-friendly software, preferably also open-source, we provide a list of tools designed to help the researcher manage the vast amount of data generated. We also indicate where such applications currently remain lacking. Moreover, we stress the eventual pitfalls of every step of such studies. This article is part of a Special Issue entitled: Computational Proteomics in the Post-Identification Era. Guest Editors: Martin Eisenacher and Christian Stephan.acceptedVersio
Extractor for ESI quadrupole TOF tandem MS data enabled for high throughput batch processing
BACKGROUND: Mass spectrometry based proteomics result in huge amounts of data that has to be processed in real time in order to efficiently feed identification algorithms and to easily integrate in automated environments. We present wiff2dta, a tool created to convert MS/MS data obtained using Applied Biosystem's QStar and QTrap 2000 and 4000 series. RESULTS: Comparing the performance of wiff2dta with the standard tools, we find wiff2dta being the fastest solution for extracting spectrum data from ABIs raw file format. wiff2dta is at least 10% faster than the standard tools. It is also capable of batch processing and can be easily integrated in high throughput environments. The program is freely available via , and is also available from Applied Biosystems. CONCLUSIONS: wiff2dta offers the possibility to run as stand-alone application or within a batch process as command-line tool integrated in automation and high-throughput environments. It is more efficient than the state-of-the-art tools provided
Future perspectives on in-vitro diagnosis of drug allergy by the lymphocyte transformation test
Acknowledgement We thank the European fund for regional development (EFRE), the German Federal State North Rhine-Westphalia (LeitmarktAgentur.NRW), Federal Institute for Drugs and Medical Devices (BfArM), and the Leibniz Institute for Analytical Sciences - ISAS-e.V. for the research project grant. The position of A.F. is financed by the research grant. Funding source The manuscript was written in context with a study related to the improvement of the lymphocyte transformation test which was funded by the European Fund for Regional Development (EFRE) and the German Federal State North Rhine-Westphalia (LeitmarktAgentur.NRW) (funding number: EFRE-0801755) and from own resources of the Federal Institute for Drugs and Medical Devices (BfArM) and the Leibniz Institute for Analytical Sciences - ISAS-e.V., Dortmund, Germany.Peer reviewedPublisher PD
Malignant transformation in a defined genetic background: proteome changes displayed by 2D-PAGE
<p>Abstract</p> <p>Background</p> <p>Cancer arises from normal cells through the stepwise accumulation of genetic alterations. Cancer development can be studied by direct genetic manipulation within experimental models of tumorigenesis. Thereby, confusion by the genetic heterogeneity of patients can be circumvented. Moreover, identification of the critical changes that convert a pre-malignant cell into a metastatic, therapy resistant tumor cell, however, is one necessary step to develop effective and selective anti-cancer drugs. Thus, for the current study a cell culture model for malignant transformation was used: Primary human fibroblasts of the BJ strain were sequentially transduced with retroviral vectors encoding the genes for hTERT (cell line BJ-T), simian virus 40 early region (SV40 ER, cell line BJ-TE) and H-Ras V12 (cell line BJ-TER).</p> <p>Results</p> <p>The stepwise malignant transformation of human fibroblasts was analyzed on the protein level by differential proteome analysis. We observed 39 regulated protein spots and therein identified 67 different proteins. The strongest change of spot patterns was detected due to integration of SV40 ER. Among the proteins being significantly regulated during the malignant transformation process well known proliferating cell nuclear antigen (PCNA) as well as the chaperones mitochondrial heat shock protein 75 kDa (TRAP-1) and heat shock protein HSP90 were identified. Moreover, we find out, that TRAP-1 is already up-regulated by means of SV40 ER expression instead of H-Ras V12. Furthermore Peroxiredoxin-6 (PRDX6), Annexin A2 (p36), Plasminogen activator inhibitor 2 (PAI-2) and Keratin type II cytoskeletal 7 (CK-7) were identified to be regulated. For some protein candidates we confirmed our 2D-PAGE results by Western Blot.</p> <p>Conclusion</p> <p>These findings give further hints for intriguing interactions between the p16-RB pathway, the mitochondrial chaperone network and the cytoskeleton. In summary, using a cell culture model for malignant transformation analyzed with 2D-PAGE, proteome and cellular changes can be related to defined steps of tumorigenesis.</p
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Malignant transformation in a defined genetic background: Proteome changes displayed by 2D-PAGE
Background: Cancer arises from normal cells through the stepwise accumulation of genetic alterations. Cancer
development can be studied by direct genetic manipulation within experimental models of tumorigenesis.
Thereby, confusion by the genetic heterogeneity of patients can be circumvented. Moreover, identification of the
critical changes that convert a pre-malignant cell into a metastatic, therapy resistant tumor cell, however, is one
necessary step to develop effective and selective anti-cancer drugs. Thus, for the current study a cell culture model
for malignant transformation was used: Primary human fibroblasts of the BJ strain were sequentially transduced
with retroviral vectors encoding the genes for hTERT (cell line BJ-T), simian virus 40 early region (SV40 ER, cell line
BJ-TE) and H-Ras V12 (cell line BJ-TER).
Results: The stepwise malignant transformation of human fibroblasts was analyzed on the protein level by
differential proteome analysis. We observed 39 regulated protein spots and therein identified 67 different proteins.
The strongest change of spot patterns was detected due to integration of SV40 ER. Among the proteins being
significantly regulated during the malignant transformation process well known proliferating cell nuclear antigen
(PCNA) as well as the chaperones mitochondrial heat shock protein 75 kDa (TRAP-1) and heat shock protein HSP90
were identified. Moreover, we find out, that TRAP-1 is already up-regulated by means of SV40 ER expression
instead of H-Ras V12. Furthermore Peroxiredoxin-6 (PRDX6), Annexin A2 (p36), Plasminogen activator inhibitor 2
(PAI-2) and Keratin type II cytoskeletal 7 (CK-7) were identified to be regulated. For some protein candidates we
confirmed our 2D-PAGE results by Western Blot.
Conclusion: These findings give further hints for intriguing interactions between the p16-RB pathway, the
mitochondrial chaperone network and the cytoskeleton. In summary, using a cell culture model for malignant
transformation analyzed with 2D-PAGE, proteome and cellular changes can be related to defined steps of
tumorigenesi
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Why phosphoproteomics is still a challenge
Despite continuous improvements phosphoproteomics still faces challenges that are often neglected,
e.g. partially poor recovery of phosphopeptide enrichment, assessment of phosphorylation
stoichiometry, label-free quantification, poor behavior during chromatography, and general limitations of
peptide-centric proteomics. Here we critically discuss current limitations that need consideration in both
qualitative and quantitative studies
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DipA, a pore-forming protein in the outer membrane of lyme disease spirochetes exhibits specificity for the permeation of dicarboxylate
Lyme disease Borreliae are highly dependent on the uptake of nutrients provided by their hosts. Our study describes the
identification of a 36 kDa protein that functions as putative dicarboxylate-specific porin in the outer membrane of Lyme
disease Borrelia. The protein was purified by hydroxyapatite chromatography from Borrelia burgdorferi B31 and designated
as DipA, for dicarboxylate-specific porin A. DipA was partially sequenced, and corresponding genes were identified in the
genomes of B. burgdorferi B31, Borrelia garinii PBi and Borrelia afzelii PKo. DipA exhibits high homology to the Oms38 porins
of relapsing fever Borreliae. B. burgdorferi DipA was characterized using the black lipid bilayer assay. The protein has a singlechannel
conductance of 50 pS in 1 M KCl, is slightly selective for anions with a permeability ratio for cations over anions of
0.57 in KCl and is not voltage-dependent. The channel could be partly blocked by different di- and tricarboxylic anions.
Particular high stability constants up to about 28,000 l/mol (in 0.1 M KCl) were obtained among the 11 tested anions for
oxaloacetate, 2-oxoglutarate and citrate. The results imply that DipA forms a porin specific for dicarboxylates which may
play an important role for the uptake of specific nutrients in different Borrelia species
compomics-utilities: an open-source Java library for computational proteomics
<p>Abstract</p> <p>Background</p> <p>The growing interest in the field of proteomics has increased the demand for software tools and applications that process and analyze the resulting data. And even though the purpose of these tools can vary significantly, they usually share a basic set of features, including the handling of protein and peptide sequences, the visualization of (and interaction with) spectra and chromatograms, and the parsing of results from various proteomics search engines. Developers typically spend considerable time and effort implementing these support structures, which detracts from working on the novel aspects of their tool.</p> <p>Results</p> <p>In order to simplify the development of proteomics tools, we have implemented an open-source support library for computational proteomics, called compomics-utilities. The library contains a broad set of features required for reading, parsing, and analyzing proteomics data. compomics-utilities is already used by a long list of existing software, ensuring library stability and continued support and development.</p> <p>Conclusions</p> <p>As a user-friendly, well-documented and open-source library, compomics-utilities greatly simplifies the implementation of the basic features needed in most proteomics tools. Implemented in 100% Java, compomics-utilities is fully portable across platforms and architectures. Our library thus allows the developers to focus on the novel aspects of their tools, rather than on the basic functions, which can contribute substantially to faster development, and better tools for proteomics.</p
Disentangling thermal stress responses in a reef-calcifier and its photosymbionts by shotgun proteomics
This project was funded by the Leibniz Association (SAW-2014-ISAS-2) awarded to AS and HW and supported by the Ministerium für Kultur und Wissenschaft des Landes Nordrhein-Westfalen, the Regierende Bürgermeister von Berlin - inkl. Wissenschaft und Forschung, and the Bundesministerium für Bildung und Forschung. Sampling was conducted under the Research Permit No. FKNMS-2015–026, issued to Pamela Hallock who is warmly acknowledged for her general support and assistance during fieldwork.Peer reviewedPublisher PD
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