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.
Wåhlander, Åsa, et al.
(författare)
Parallel post-source decay for increasing protein identification confidence levels from 2-D gels
2008
Ingår i: Proteomics. - : Wiley. - 1615-9853 .- 1615-9861. ; 8:9, s. 1771-1779
Tidskriftsartikel (refereegranskat) abstract
Peptide mass fingerprinting (PMF) has over the years become one of the most commonly used tools for high-throughput analysis and identification of proteins. This method is applicable when relatively simple samples have to be analysed and it is commonly used for analysing proteins previously separated by 2-DE. The most common type of instrument used for this approach is the MALDI-TOF that has proved to be particularly suitable for the PMF analysis because of its characteristics of speed, robustness, sensitivity and automation. We have used a MALDI-TOF equipped with a novel parallel PSD capability (MALDI micro MX), to perform the analysis of two sets of different biological samples isolated by 2-DE. By using a method that integrates the data obtained by PMF analysis with the PSD data obtained in the same experiment, we show that the new multiplexed PSD solution increases the protein identification rate compared to the normal PMF approach. We also investigated the use of a charge-directed fragmentation modification reagent to improve the identification rate and confidence levels.
3.
Andersson, Tomas, et al.
(författare)
Automating MALDI Sample Plate Loading
2007
Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 6:2, s. 894-896
Tidskriftsartikel (refereegranskat) abstract
We describe the design and implementation of a generic robotic solution to automate the loading of MALDI sample plates into a mass spectrometer. The soft- and hardware aspects are described together with the various safety issues that need to be addressed. The automation increases thoughput by a factor of between 5- and 80-fold.
4.
Berggård, Tord, et al.
(författare)
Methods for the detection and analysis of protein-protein interactions
2007
Ingår i: Proteomics. - : Wiley. - 1615-9861 .- 1615-9853. ; 7:16, s. 2833-2842
Forskningsöversikt (refereegranskat) abstract
A large number of methods have been developed over the years to study protein-protein interactions. Many of these techniques are now available to the nonspecialist researcher thanks to new affordable instruments and/or resource centres. A typical protein-protein interaction study usually starts with an initial screen for novel binding partners. We start this review by describing three techniques that can be used for this purpose: (i) affinity-tagged proteins (ii) the two-hybrid system and (iii) some quantitative proteomic techniques that can be used in combination with, e.g., affinity chromatography and coimmunoprecipitation for screening of protein-protein interactions. We then describe some public protein-protein interaction databases that can be searched to identify previously reported interactions for a given bait protein. Four strategies for validation of protein-protein interactions are presented: confocal microscopy for intracellular colocalization of proteins, coimmunoprecipitation, surface plasmon resonance (SPR) and spectroscopic studies. Throughout the review we focus particularly on the advantages and limitations of each method.
5.
Bäck, Peter, et al.
(författare)
Automating gel image acquisition
2003
Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 2:6, s. 662-664
Tidskriftsartikel (refereegranskat) abstract
We describe the design and implementation of a robotic solution to automate the acquisition of gel images. The soft- and hardware aspects are outlined together with the various safety aspects that need to be addressed.
6.
Corthals, Garry L., et al.
(författare)
The transition of the European Proteomics Association into the future
2011
Ingår i: Journal of Proteomics. - : Elsevier BV. - 1874-3919. ; 75:1, s. 18-22
Tidskriftsartikel (övrigt vetenskapligt/konstnärligt) abstract
The following report provides an overview of the discussions and outcome of the EuPA General Council meeting that took place in Estoril 20-21 October 2010. During the annual meeting future policy and action plans in a variety of areas are decided. Several important points were decided upon during this meeting including the expansion of the EuPA Executive Committee by introducing a new EuPA committee - EuPA Developments - that will initially spearhead activities in standardisation, imaging ms and biobanking. The EuPA General Council also invited Russia as its 17th member. More details about these and additional activities are presented in the article. (C) 2011 Published by Elsevier B.V.
7.
8.
James, Peter, et al.
(författare)
The International Proteomics Tutorial Programme - reaching out to the next generation proteome scientists
2011
Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 10:8, s. 3311-3312
Tidskriftsartikel (refereegranskat) abstract
One of the most critical functions of the various Proteomics organizations is the training of young scientists and the dissemination of information to the general scientific community. The education committees of the Human Proteome Organisation (HUPO) and the European Proteomics Association (EuPA) together with the other local proteomics associations are therefore launching a joint Tutorial Program to meet these needs. The level is aimed at Masters/PhD level students with good basic training in biology, biochemistry, mathematics and statistics. The Tutorials will consist of a review/teaching article with an accompanying talk slide presentation for classroom teaching. The Tutorial Program will cover core techniques and basics as an introduction to scientists new to the field. The entire series of articles and slides will be made freely available for teaching use at the Journals and Organizations homepages.
9.
Bruzelius, Katharina, et al.
(författare)
Biosynthesis of selenoproteins in cultured bovine mammary cells.
2008
Ingår i: Journal of Trace Elements in Medicine and Biology. - : Elsevier BV. - 1878-3252 .- 0946-672X. ; 22:3, s. 224-233
Tidskriftsartikel (refereegranskat) abstract
The biosynthesis of selenoproteins was studied in relation to milk formation and mammary cell biology by incubating the bovine mammary cell line MAC-T with (75Se)selenite. Intracellular proteins and proteins secreted into the cell culture medium were separated by 2D electrophoresis, the selenoproteins were detected by autoradiography, and the proteins were identified by MALDI-TOF. Approximately 35 75Se-containing spots were found in the cell proteins from MAC-T cells. Among them, one-third showed high intensity. The strongest spot was identified as glutathione peroxidase 1. About 20 spots were observed in protein precipitated from cell culture medium, one-third of them being distinctly visible. In an attempt to study a perturbation of the system, the effect of retinoic acid (RA) on the formation of selenoproteins was investigated. The concentration of 75Se in total cell protein was reduced by about 35% in cells cultured with RA compared with control cells, while the opposite effect was observed in protein precipitated from cell culture medium, which contained 60% more 75Se in RA-treated samples than in controls. There were also indications that RA might affect different selenoproteins in different ways. The methods described provide a promising approach for further studies of the regulation of selenoprotein formation in the mammary gland.
10.
Levander, Fredrik, et al.
(författare)
Automated methods for improved protein identification by peptide mass fingerprinting
2004
Ingår i: Proteomics. - Weinheim : Wiley. - 1615-9861 .- 1615-9853. ; 4:9, s. 2594-2601
Tidskriftsartikel (refereegranskat) abstract
In order to maximize protein identification by peptide mass fingerprinting noise peaks must be removed from spectra and recalibration is often required. The preprocessing of the spectra before database searching is essential but is time-consuming. Nevertheless, the optimal database search parameters often vary over a batch of samples. For high-throughput protein identification, these factors should be set automatically, with no or little human intervention. In the present work automated batch filtering and recalibration using a statistical filter is described. The filter is combined with multiple data searches that are performed automatically. We show that, using several hundred protein digests, protein identification rates could be more than doubled, compared to standard database searching. Furthermore, automated large-scale in-gel digestion of proteins with endoproteinase LysC, and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis, followed by subsequent trypsin digestion and MALDI-TOF analysis were performed. Several proteins could be identified only after digestion with one of the enzymes, and some less significant protein identifications were confirmed after digestion with the other enzyme. The results indicate that identification of especially small and low-abundance proteins could be significantly improved after sequential digestions with two enzymes.
