Laboratory for Biochemistry and Proteomics
Institute of Molecular Biology
- Pseudorabies Virus
- Herpes Simplex Virus-1
- Bovine Herpesvirus-1
- Influenza A Virus
Application of proteomics in virology
In recent years, proteome analysis has been introduced and successfully applied to virtually all fields of the life sciences, including virology. The combination of high-resolution protein analysis with mass spectrometry allows the qualitative and quantitative assay of hundreds or even thousands of proteins that characterise a specific state of a cell, e.g. after virus infection. The Laboratory of Biochemistry and Proteomics is equipped with large-size two-dimensional gel electrophoresis equipment, a robot for automatic in-gel digestion of proteins, and an Ultraflextreme TOF/TOF MALDI mass spectrometer coupled to a nano-LC. Quantitative modulations in the proteome composition and post-translational modifications are characteristic for the response of animal cells to stimuli. These alterations can be monitored in detail by the ‚stable isotope labelling with amino acids in cell culture’ (SILAC) procedure (Ong et al., 2002), which we have applied to screen for hitherto unknown reactions of target cells to an infection with Pseudorabies Virus, a herpesvirus, (Skiba et al, 2008). The SILAC approach was also used to compare the proteome of target cells after infection with wild type PrV and a deletion mutant (Skiba et al., 2010) to study the impact of a single viral protein on the proteome of an infected cell.
Ramp K, Skiba M, Karger A, Mettenleiter TC, Römer-Oberdörfer A. 2011. Influence of insertion site of the avian influenza virus haemagglutinin (HA) gene within the Newcastle disease virus genome on HA expression. J Gen Virol 92:355-360.
Skiba M, Glowinski F, Koczan D, Mettenleiter TC, Karger A. (2010). Gene expression profiling of Pseudorabies virus (PrV) infected bovine cells by combination of transcript analysis and quantitative proteomic techniques. Vet Microbiol 143:14-20.
Skiba M, Mettenleiter TC, Karger A. (2008). Quantitative whole-cell proteome analysis of pseudorabies virus-infected cells. J Virol 82:9689-99.
Composition of mutated Pseudorabies Virus particles
The Pseudorabies Virus (PrV) genome codes for over 70 proteins, many of which are also incorporated into the virus particle. Virions also contain constituents of cellular origin e.g. the viral lipid membrane and cellular proteins, which may have a so far unknown function for the viral life cycle. Highly purified Pseudorabies virus particles were analysed by two-dimensional gel electrophoresis and a 2D-map of PrV was established. Moroeover, mass spectrometric quantification (‚stable isotope labelling with amino acids in cell culture’, SILAC) of PrV structural proteins in wild type and mutated virions identified new interactions between the constituents of PrV particles (Michael et al., 2006; Michael et al., 2006; Böttcher et al., 2006; Michael et al., 2007; Fuchs et al., 2007). Ongoing projects focus on the analysis of the composition of morphogenetic intermediates of herpes virions.
Fuchs W, Granzow H, Klupp BG, Karger A, Michael K, Maresch C, Klopfleisch R, Mettenleiter TC. (2007). Relevance of the interaction between alphaherpesvirus UL3.5 and UL48 proteins for virion maturation and neuroinvasion. J Virol 81:9307-18.
Michael K, Klupp BG, Karger A, Mettenleiter TC. (2007). Efficient incorporation of tegument proteins pUL46, pUL49, and pUS3 into pseudorabies virus particles depends on the presence of pUL21. J Virol 81:1048-51.
Michael K, Böttcher S, Klupp BG, Karger A, Mettenleiter TC. (2006). Pseudorabies virus particles lacking tegument proteins pUL11 or pUL16 incorporate less full-length pUL36 than wild-type virus, but specifically accumulate a pUL36 N-terminal fragment. J Gen Virol 87:3503-7.
Böttcher S, Klupp BG, Granzow H, Fuchs W, Michael K, Mettenleiter TC. (2006). Identification of a 709-amino-acid internal nonessential region within the essential conserved tegument protein (p)UL36 of pseudorabies virus. J Virol 80:9910-5.
Michael K, Klupp BG, Mettenleiter TC, Karger A. (2006). Composition of pseudorabies virus particles lacking tegument protein US3, UL47, or UL49 or envelope glycoprotein E. J Virol 80:1332-9.
Mass spectrometric diagnosis of Influenza A Virus-infections
The outbreak of bird flu in 2005 has emphasized the need for rapid and reliable high-throughput diagnosis of Influenza A Virus (AIV) infections. We have recently developed such a method on the basis of MALDI mass spectrometry (Michael, Harder et al., 2009). First step is the amplification of a specific region of the hemagglutinin (HA) gene spanning the cleavage site of the HA protein, which determines the pathogenicity of the virus. The PCR product is then digested with a dedicated restriction enzyme cocktail and the resulting fragments are analysed by mass spectrometry (‘restriction fragment mass fingerprint’, RFMF). Analysis of the spectra allowed even the differentiation of different AIV strains of the same subtype H5N1. In cooperation with the FLI’s workgroup biomathematics the theoretic foundations for classification of PCR products by MALDI-TOF MS are investigated (Ziller et al., 2011).
Ziller M, Gussmann M, Karger A. 2011. Mathematical Aspects of Classifying Mass Spectra. Diagnostic Discrimination of Influenza-subtypes. In: Dress A et al. (eds.), The Math of Flu, Heft 17, Reihe: Biometrie und Medizinische Information – Greifswalder Seminarberichte, Shaker Verlag, Aachen, ISBN: 978-3-8322-9763-3, pp. 101-123.
Michael K, Harder TC, Mettenleiter TC, Karger A. (2009). Diagnosis and strain differentiation of avian influenza viruses by restriction fragment mass analysis. J Virol Methods 158:63-9.
MALDI-typing of bacteria and animal tissue cultures
Identification and taxonomic classification of bacteria by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has become increasingly popular in bacteriological laboratories as ‘intact cell mass spectrometry’ (ICMS). In addition to the low cost and high speed of analysis, MALDI-typing in some cases can be a valuable complementation to classical bacteriological procedures, e.g. in cases where sub-species differentiation of bacteria is required.
MALDI-typing relies on the comparison of the sample spectrum with reference spectra from a commercial or an in-house database. Except for an authentic reference spectrum, no additional data (e.g. sequence data) of the organism to be identified is required. For bacteriological routine, our laboratory offers the identification of bacterial samples with a database containing over 3500 reference spectra, part of which is dedicated to veterinary medicine. Intact cell MALDI-spectra can also be used for phylogenetic or epidemiological studies.
Together with the FLI’s cell culture repository (Collection of Cell Lines in Veterinary Medicine, CCLV) a protocol for the characterization of cultured animal cells by ICMS was developed (Karger et al., 2010) which allows the species determination of cell cultures of unknown or doubtable origin. This technology is also applicable for characterization of ticks, which was shown in cooperation with the NRL for tick-borne diseases (Karger et al., 2012a).
Karger A, Kampen H, Bettin B, Dautel H, Ziller M, Hoffmann B, Süss J, Klaus C. 2012a. Species determination and characterization of developmental stages of ticks by whole-animal matrix-assisted laser desorption/ionization mass spectrometry. Ticks Tick-Borne Dis 3:78-89.
Karger A, Stock R, Ziller M, Elschner MC, Bettin B, Melzer F, Maier T, Kostrzewa M, Scholz HC, Neubauer H, Tomaso H. 2012b. Rapid identification of Burkholderia mallei and Burkholderia pseudomallei by intact cell Matrix-assisted Laser Desorption/Ionisation mass spectrometric typing. BMC Microbiol 12:229.
Löhr CV, Polster U, Kuhnert P, Karger A, Rurangirwa FR, Teifke JP. 2012. Mesenteric Lymphangitis and Sepsis Due to RTX Toxin-Producing Actinobacillus spp in 2 Foals With Hypothyroidism-Dysmaturity Syndrome. Vet Pathol 49:592-601.
Karger, A., M. Ziller, B. Bettin, B. Mintel, S. Schares, and L. Geue. (2011). Determination of Serotypes of Shiga Toxin-Producing Escherichia coli Isolates by Intact Cell Matrix-Assisted Laser Desorption Ionization – Time of Flight Mass Spectrometry. Appl Environ Microbiol 77:896-905.
Karger A, Bettin B, Lenk M, Mettenleiter TC. (2010). Rapid characterisation of cell cultures by matrix-assisted laser desorption/ionisation mass spectrometric typing. J Virol Methods 164:116-21.
Mass spectrometry service
The Laboratory for Biochemistry and Proteomics offers assistance and advice for all FLI staff in questions of protein analytics concerning
- identification of proteins by ‚peptide mass fingerprint’ (Ultraflex TOF/TOF mass spectrometer, Bruker)
- N- and C-terminal protein sequencing by (ISD spectra)
- two-dimensional gel electrophoresis, also large-size (DodecaCell, BioRad)
- ‚off gel’ protein or peptide focusing (Fractionator 3200, Agilent)
- identification of bacteria