Peter Højrup

Peter Højrup graduated from Aarhus University, Department of Molecular Biology. He then went on and obtained a Lic. Scient. degree (Ph.D.) from Odense University (now University of Southern Denmark), Department of Molecular Biology, in the emerging field of protein mass spectrometry under the supervision of Prof. Peter Roepstorff. The main focus of the study was development of new methods for primary structure elucidatin by coupling classical protein chemical methods with mass spectrometry. After a couple of years as a postdoc at the University of Aarhus, he returned to Odense where he rejoined Peter Roepstorff's group. In 1991 he became associate professor at Department of Biochemistry and Molecular Biology, University of Southern Denmark, where he since 2008 has been professor MSO.

Head of research: Professor wsr Peter Højrup

Research group:
PhD fellow Anette Holck Draborg (co-supervisor)
PhD fellow Søren Heissel
PhD fellow Sheila Lefoli Maibom-Thomsen

PhD student Jakub Zbigniew Kaczmarek

Laboratory technician trainee Andreea Teodora Bejenarieu


Structure and function of proteins

Proteins are by and large the working molecules of life, and the understanding of their structure, is essential for understanding how they function in the body. The tendency over recent years of medical treatment being not just by small molecules, but increasingly by proteins manufactured by recombinant techniques, has put increasing pressure on techniques to perform a complete characterization of a given protein.
Our research for the last several years has mainly been concentrated on developing new methods for structure determination of proteins and applying these methods to proteins related to immunity, both from the innate and the adaptive system. Being extracellular, these proteins are often extensively post-translationally modified, and although many of them have been studied for several years, there are still many surprises hidden in their structure.

Current research projects

Protein structure through chemical cross-linking and mass spectrometry
The three-dimensional structure of proteins is traditionally carried out by x-ray diffraction or NMR. Both methods determine structures with high resolution, but suffer from a number of draw-backs. The protein has to be well ordered and for x-ray diffraction you need to have the protein crystallized, for NMR there is a size limit and the quantities needed are quite large. 
Chemical cross-linking of proteins has the potential of determining the overall structure of a protein in a fast and sensitive way. The resolution of the resulting structure is very low, and has to be combined with modeling of homologous structures to yield detailed results. However, the method can be used on simple mixtures; it is quite sensitive and can also determine sites of interaction if multiple proteins participate in a complex. The main limitation is that the number of cross-links made is not high and very sensitive methods, both in mass spectrometry and in bioinformatics, is needed for successful results.  Over recent years we have made great advances in the technique, both with regards to mass spectrometry and, most importantly, with regard to bioinformatics. The technology is now mature enough to be used for biological studies, and we are applying it to various proteins involved in the innate and adaptive immunity.

Structure and function of calreticulin

Calreticulin is an essential protein which is almost exclusively located in the endoplasmatic reticulum where it is the protein present in the highest concentration. The protein binds monoglucosylated N-linked carbohydrates and functions as a chaperone in the folding pathway of most secreted proteins. In addition it functions in the calcium homeostasis with a high capacity, and it is further part of the peptide loading complex, ensuring that MHC is correctly loaded with a suitable peptide.
We have shown that in addition to carbohydrates, calreticulin also has a strong peptide binding ability, and can thus bind non-glycosylated proteins. Furthermore, we have shown how it interacts with a number of other proteins, in particular immunoglobulins and a number of proteins in the innate immunity. Structurally the protein is very unusual with a beta-barrel as a body, an extended arm stabilized by beta-sheets and an extended 50-residue C-terminal of unknown structure. We have previously shown that it easily multimerizes, is quite mobile, and in many circumstances does not seem to be structually stable.
In our current research we are investigating an observation that very small changes in temperature seems to lead to significant changes in the protein conformation, which affects function and activity. Furthermore, we have observed activities that indicate a functionality not previously described. In collaboration with Statens Serum Institut and researchers abroad, we are now making a myriad of variants in order to determine the specific structural changes and the chemistry defining the newly determined activities.

High-sensitivity lc-ms/ms analysis for increasing vaccine safety
By far the most vaccines being developed today are recombinant proteins that are produced through gene-modified non-human cell lines. Although all traces of the host cell should ideally be removed during the purification of the vaccine product, some host cell proteins will invariably be co-purified. Remains of these host cell proteins, even at the ppm level, presents a latent risk of a serious immune response in the recipient. Because of this authorities puts an increased pressure on vaccine producers to map the exact content of the final vaccine. 
The current routine state of the art methods for identifying these impurities are not particularly sensitive, which the current project aims to improve considerably. In a collaboration with Statens Serum Institute (vaccine producer) and Alphalyse A/S (commercial protein analysis company) we will develop a routine method with a 10-100 times increased sensitivity towards detection of host cell proteins in recombinant protein pharmaceuticals. Making the method both fast and sensitive will give the pharmaceutical companies further possibilities to improve their purification methods at a much earlier stage than is currently the standard, and will thus increase vaccine safety.

Selected publications

Probing the structure of human protein disulfide isomerase by chemical cross-linking combined with mass spectrometry
Peng, L, Rasmussen, MI, Chailyan, A, Houen, G & Højrup, P, , J. Proteom, 2014. 108, 1-16

Site-specific glycoprofiling of N-linked glycopeptides using MALDI-TOF MS
Thaysen-Andersen, M, Mysling, S, and Højrup, P: , Anal. Chem. 2009, 81(10):3933-43.

CrossWork: Software-assisted Identification of Cross-linked Peptide
Morten I Rasmussen, Jan C. Refsgaard, Li Peng, Gunnar Houen & Peter Højrup, J. of Proteomics. 2011, 74, 1871-83

A full list of publications by professor wsr Peter Højrupcan be found here.

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