Proteomics Core Facility at Rikshospitalet HF
Proteome Core Facility at OUH-Rikshospitalet
The Proteomics Core Facility (PCF) at Oslo University Hospital – Rikshospitalet provides proteomic service to all research groups in the HSØ region in need of it. We are constantly expanding our service portfolio to serve the increasing demands for such analysis. New techniques and expertise are now available as service. A new group leader, Gustavo de Souza, was recently hired to lead the PCF.
Recently, former group leader of PCF Burkhard Fleckenstein left the University of Oslo to follow his career in Germany. For his position, the Institute of Immunology hired Gustavo de Souza, a 33 years-old researcher with 9 years of experience in mass spectrometry and 8 years of experience in proteomics. Gustavo de Souza has a PhD in Cell Biology from the University of Sao Paulo in 2004, and after that he had two and half years of post-doctoral work at the Max-Planck Institute fur Biochemie, Martinsried, Germany. At that Institute he worked with Matthias Mann, one of the leading scientists in the field of Proteomics. In addition to expertise with the instruments currently available at the PCF, Gustavo de Souza has also knowledgeable experience with advanced instrumentation such as the LTQ-Orbitrap, and bioinformatic skills using MaxQuant for SILAC labeling and label-free quantitation analysis. Scientifically, Gustavo de Souza has a competent CV with 24 publications (23 using mass spectrometry), 14 of those in the year of 2009-2010 alone.
In addition, in 2010, the PCF had further extended its proteomic service provided to biomedical research groups in the HSØ region. The number of data analysis had surpassed 400 samples from over 36 different groups, which is as much as the total number of analysis performed in 2008 and 2009 all together. This clearly demonstrates that the PCF is a well accepted institution within the research community. And also indicates the increase in service demand that the PCF is now facing, specially regarding analysis of complex protein mixtures using Electrospray Ionization (ESI) instruments. However, it is worthy mentioning that two of three of PCF instruments are about to complete 5 years of usage, which is considered by most of the mass spectrometers manufacturers the optimal life-time of such instruments. Therefore, it is safe to assume that the PCF not only faces an increase in service demand while also faces an expected reduction in instrument performance in 2011-2012.
The ion trap mass spectrometer installed in Sept’09 which was cofunded by HSØ had severe downtime in 2010 due to troubleshooting with its liquid chromatography system. However the instrument has been stable in the last semester of the year, and since then it has been used on routine analysis more often. We are currently optimizing it to characterize phosphorylation and glycosylation in peptides. We will be soon offering such type of analysis to the community, and currently we already have the first samples being processed for characterization of glycopeptides from a group within Rikshospitalet. This is in accordance with the aims proposed in this application.
New methods have been developed and published, focusing mainly in peptide/protein pull-downs. Those helped the researchers involved in PCF to elucidate that transglutaminase 2 affinity to gluten favors celiac disease epitopes (Dørum et al., 2010), as well as binding signatures for transglutaminase 3 and 6 (Stamnaes et al., 2010). Mass spectrometry was also used to support the design of high-affinity peptides for antigen DQ2 (Jüse et al., 2010). Finally, a new method for enrichment of citrulline-containing peptides, which are important markers in rheumatoid arthritis, was also developed (Tutturen et al., 2010).
The PCF is also a node in the national-wide proteomics consortium NorProteomics (FUGE/NFR). Together with the proteomic laboratories at the Biotechnology Centre in Oslo (Bernd Thiede) and UMB at Ås (Vincent Eijsink), the PCF has received funding from FUGE-Øst for an expert in bioinformatics. This position is shared between laboratories, and Magnus Arntzen was hired for it. He was formerly an engineer at the Proteomic Unit of Bergen, and had bioinformatic collaboration with PCF current leader Gustavo de Souza (this collaboration resulted in two publications in journals ‘Bioinformatics’ and ‘Molecular and Cellular Proteomics’ in 2010). Since his arrival Magnus Arntzen has been an active member in all groups and results are already being published (Dørum et al., 2010; Arntzen et al., 2011).
Proteomics Core Facility at OUH-Rikshospitalet
The Proteomics Core Facility (PCF) at Oslo University Hospital-Rikshospitalet provides proteomic service to all research groups in the HSØ region in need of it. We are constantly expanding our service portfolio to serve the increasing request for such analyses. New techniques were published and are now available as service.
In 2009, the PCF has further extended its proteomics service provided to biomedical research groups in the HSØ region. The number of users was again increasing and a broad scale of proteomics analyses, including newly developed techniques, was offered. A new liquid chromatography-ion trap mass spectrometer (equipped with electron transfer dissociation, ETD), which was cofunded by HSØ, was installed in Sept’09. This instrumentation allows new types of analysis (with the focus on posttranslational modifications of proteins) and enables us to cope with the still increasing number of requested analyses. Altogether, the PCF is a well accepted institution within the research community of HSØ.
In accordance with the aims presented in the application, several new technologies have been further developed on a collaborative basis and resulted in publications, e.g. the analysis of protein phosphorylation (Øverbye and Seglen 2009), quantitative proteomic analysis of enzymatic deamidation (Dørum at al, 2009) or a new targeted approach to study protein citrullination (Stensland, Holm et al, 2009). The equipment and know-how present at the PCF has been essential in further studies on celiac disease (Fallang et al, 2009; Jüse et al, 2009), rheumatoid arthritis (Stensland, Pollmann et al, 2009), or Neisseria meningitis (Lång et al, 2009).
As described in Project 1 of the application (“High throughput affinity proteomics”), a strong collaboration has been established to Fridtjof Lund-Johansen at our institute. This project which focuses on the development of antibody array technology has nicely progressed (Wu et al, Mol Cell Proteomics, 2009) and is combined with protein fractionation and mass spectrometric methodology available at the PCF.
In Project 2, the PCF and Andre Ingebretsen (Institute of Microbiology, Rikshospitalet) have together setup a methodology for the rapid identification of clinical relevant bacteria and fungi by use of the PCF’s MALDI-TOF mass spectrometer. After the approach has been established at the PCF, the Institute of Microbiology has acquired an own instrument and performs today a large number of analyses by itself. This project has been an excellent example of know-transfer from the PCF to another institute at a HSØ hospital.
The PCF is also a node in the national-wide proteomics consortium NorProteomics (established by FUGE/NFR). Together with the proteomics laboratories at the Biotechnology Centre in Oslo (Bernd Thiede) and UMB at Ås (Vincent Eijsink), the PCF has received funding from FUGE-Øst for an expert in bioinformatics. This position is shared between the three laboratories.
The preferred substrates for transglutaminase 2 in a complex wheat gluten digest are Peptide fragments harboring celiac disease T-cell epitopes.
PLoS One 2010;5(11):e14056. Epub 2010 nov 19
Gluten T cell epitope targeting by TG3 and TG6; implications for dermatitis herpetiformis and gluten ataxia.
Amino Acids 2010 Nov;39(5):1183-91. Epub 2010 mar 19
Redox regulation of transglutaminase 2 activity.
J Biol Chem 2010 Aug;285(33):25402-9. Epub 2010 jun 14
A technique for the specific enrichment of citrulline-containing peptides.
Anal Biochem 2010 Aug;403(1-2):43-51. Epub 2010 apr 23
Design of new high-affinity peptide ligands for human leukocyte antigen-DQ2 using a positional scanning peptide library.
Hum Immunol 2010 May;71(5):475-81. Epub 2010 feb 6
Soluble HLA-DQ2 expressed in S2 cells copurifies with a high affinity insect cell derived protein.
Immunogenetics 2009 Feb;61(2):81-9. Epub 2008 nov 6
Primary sequence, together with other factors, influence peptide deimination by peptidylarginine deiminase-4.
Biol Chem 2009 Feb;390(2):99-107.
Identification of neisserial DNA binding components.
Microbiology 2009 Mar;155(Pt 3):852-62.
A quantitative analysis of transglutaminase 2-mediated deamidation of gluten peptides: implications for the T-cell response in celiac disease.
J Proteome Res 2009 Apr;8(4):1748-55.
Targeted analysis of protein citrullination using chemical modification and tandem mass spectrometry.
Rapid Commun Mass Spectrom 2009 Sep;23(17):2754-62.
Differences in the risk of celiac disease associated with HLA-DQ2.5 or HLA-DQ2.2 are related to sustained gluten antigen presentation.
Nat Immunol 2009 Oct;10(10):1096-101. Epub 2009 aug 30
Phosphorylated and non-phosphorylated forms of catechol O-methyltransferase in rat liver, brain and other tissues.
Biochem J 2009 Jan;417(2):535-45.
Exploring peptide binding to the disease associated HLA-DQ2.5 molecule by the use of peptide libraries
- september 2010
- Ludvig M. Sollid
Substrate specificity of transglutaminases for gluten peptides
- oktober 2010
- Burkhard Fleckenstein
Investigating celiac disease using recombinant soluble MHC class II molecules
- september 2009
- Ludvig M. Sollid