High throughput protein analysis using nanoflow-liquid-chromatography-

The LC-orbitrap-MS has been applied in corporation with Prof. Dr. rer. nat. Andreas Pich, MS lab in the Institute for Toxicology, Medical School Hannover.

Peptide extracts were combined, dried and redissolved in 10 μl 2% ACN, 0.1% TFA. LC-MS/MS analysis was performed on an LTQ Orbitrap Velos mass spectrometer (Thermo Fisher Scientific) as described recently (Boer et al., 2011). Briefly, an appropriate sample amount was loaded onto a nanoflow ultra-high pressure liquid chromatography system (RSLC, Dionex) equipped with a trapping column (5 mm C18 particle, 75 mm ID, C18

material, 2 cm length, PepMap, Dionex) and separating column (2 mm C18 particle, 75 mm ID, 50 cm length, PepMap, Dionex). After trapping, peptides were eluted with a linear gradient of buffer B (80% ACN, 0.1% formic acid) in buffer A (0.1% formic acid) from 4%

to 25% in 60 min, 25%-50% in 25 min and 50%-90% in 5 min, after which the column was flushed for 10 min isocratically with 90% B and reconditioned to 4% B in 20 min. Flow rate was 250 nl/min with a column temperature of 40 °C. Peptides were ionized in the nanoESI source with 1.2 kV. Overview scans were acquired at a resolution of 60 k in a mass range of m/z 300-1600 in the orbitrap. The top 10 most intensive ions of charge two or three and a minimum intensity of 2000 were selected for CID fragmentation with a normalized collision energy of 38.0, an activation time of 10ms and an activation Q of 0.250 in the linear ion trap mass analyzer of the LTQ Orbitrap Velos. Fragmentation mass spectra were also recorded in the LTQ. The m/z values in a 10 ppm mass window of the selected ions were subsequently excluded from the fragmentation for 70 s. Data analysis was facilitated by proteome discoverer software 1.2 (Thermo Fisher Scientific, Langenselbold, Germany) and the Mascot search algorithm. Mascot was set up to search a customized database generated using the UniProt database (release 2012_03). It includes M. avium subspecies paratuberculosis K10 (NCBI Reference Sequence: NC_002944.2; 4350 genes, 4323 protein entries in UniProt), and a total of 6760 reviewed bovine protein entries (searched for Bos taurus). A false discovery rate of 0.01 and a Peptide-Score of 30 were used. Proteins were stated identified if at least two unique peptides were detected.

2.11. Criteria for protein identification and differential expression

Proteins from mucosa- and culture-derived MAP were considered as identified if they were detected at least twice by GeLC-MS or tube-gel trypsin digestion followed by MS in independent biological repeats. Alternatively, proteins were also considered as identified if isolated from 2D gels and giving a significant database match of the peptide mass fingerprint.

The proteins identified in MDCs were considered to be present also in CDCs after a single detection by GeLC-MS or tube-gel trypsin digestion followed by MS.

Proteins were considered as differentially expressed only if an at least 1.5-fold increase in expression could be observed in MDC preparations from each of the two cows in comparison to the corresponding CDCs.

Chapter 2 – Material and methods

36 2.12. Data processing and bioinformatics

LC-MS/MS raw data were processed using ProteinLynx™ Global Server (Version 2.1, Waters) by searching against the Mycobacterium species SWISS-PROT database downloaded on 25.09.2008 [99,308 entries]. The identification of bovine proteins was performed by searching against the NCBI nonredundant database as downloaded on August 16th 2006. All proteins identified in MDM or CDM as being specific for MAP were copied into separate Microsoft Excel data sheets. The mycobacterial origin of all proteins detected in MDM by a one peptide-hit only was confirmed by searching against the entire NCBI data base.

Prediction for membrane association or cytoplasmatic localisation of these proteins was performed with PSORT (http://psort.ims.u-tokyo.ac.jp/form.html). Verified datasets were organized according to their distribution in the Cluster of Orthologous Groups (COGs).

Pathway reconstruction was performed using the cellular overview tool from SRI’s pathway tools software (http://ecocyc.org/background.shtml) for proteins with a Reference Common Name (RCN). In order to obtain information possibly missed using the cellular overview tool the KEGG database was searched using MAP annotation numbers and M. tuberculosis orthologues identified by protein homology blast using the Multi-Genome Homology Comparison (Comparative Tools) of Comprehensive Microbial Resource (CMR) available at http://cmr.jcvi.org and the TB database (http://genome.tbdb.org) and NCBI.

Reference List

Boer, U., Lohrenz, A., Klingenberg, M., Pich, A., Haverich, A. & Wilhelmi, M. (2011). The effect of detergent-based decellularization procedures on cellular proteins and immunogenicity in equine carotid artery grafts. Biomaterials 32, 9730-9737.

Buettner, F. F., Bendalla, I. M., Bosse, J. T., Meens, J., Nash, J. H., Hartig, E., Langford, P. R. & Gerlach, G. F. (2009). Analysis of the Actinobacillus pleuropneumoniae HlyX (FNR) regulon and identification of iron-regulated protein B as an essential virulence factor. Proteomics 9, 2383-2398.

Choy, E., Whittington, R. J., Marsh, I., Marshall, J. & Campbell, M. T. (1998). A method for purification and characterisation of Mycobacterium avium subsp. paratuberculosis from the intestinal mucosa of sheep with Johne's disease. Vet Microbiol 64, 51-60.

Lu, X. N. & Zhu, H. N. (2005). Tube-gel digestion - A novel proteomic approach for high throughput analysis of membrane proteins. Molecular & Cellular Proteomics 4, 1948-1958.

Möbius, P., Fritsch, I., Luyven, G., Hotzel, H. & Kohler, H. (2009). Unique genotypes of Mycobacterium avium subsp. paratuberculosis strains of Type III. Vet Microbiol.

Shevchenko, A., Wilm, M., Vorm, O. & Mann, M. (1996). Mass spectrometric sequencing of proteins from silver stained polyacrylamide gels. Analytical Chemistry 68, 850-858.

Weigoldt, M., Meens, J., Doll, K., Fritsch, I., Möbius, P., Goethe, R. & Gerlach, G. F. (2011). Differential proteome analysis of Mycobacterium avium subsp. paratuberculosis grown in vitro and isolated from cases of clinical Johne's disease. Microbiology 157, 557-565.

3. Chapter:

Differential proteome analysis of Mycobacterium avium subsp.

paratuberculosis grown in vitro and isolated from cases of clinical Johne’s disease

Mathias Weigoldt¹, Jochen Meens¹, Klaus Doll², Isabel Fritsch³, Petra Möbius³, Ralph Goethe¹†, Gerald-F. Gerlach¹†‡

1 Institute for Microbiology, Department of Infectious Diseases, University of Veterinary Medicine Hannover, Hannover, Germany

2 Clinic for Ruminants and Swine (Internal Medicine and Surgery), Justus-Liebig-University, Giessen, Germany

3 Institute of Molecular Pathogenesis, Friedrich-Loeffler-Institut (Federal Research Institute for Animal Health), Jena, Germany

† These authors contributed equally to this work.

‡ Corresponding author. Current address: IVD GmbH, Heisterbergallee 12, 30453 Hannover, Germany.

Correspondence Gerald-F. Gerlach gfgerlach@gmx.de

Running title: Johne’s disease - bacterial proteins in the host Contents category: Microbial Pathogenicity

Keywords: M. avium subsp. paratuberculosis; differential protein expression; protein expression in the host; 2D DIGE; nUPLC-ESI Q-TOF-MS/MS

(Manuscript has been published in Microbiology 2011, 157, 557-565) (Online available: doi: 10.1099/mic.0.044859-0)

ABSTRACT

Bovine Johne’s disease (paratuberculosis), caused by M. avium subspecies paratuberculosis, poses a significant economic problem to the beef and dairy industry worldwide. Despite its relevance, however, pathogenesis of Johne’s disease is still only partially resolved. Since mycobacterial membrane proteins expressed during infection are likely to play an important role in pathogenesis, membrane-enriched fractions, namely mucosa-derived membranes (MDM) and culture-derived membranes (CDM), of M. avium subsp. paratuberculosis from three cows with clinical paratuberculosis were investigated. An initial analysis by two-dimensional difference gel electrophoresis (2D DIGE) and MALDI-TOF-MS analysis revealed four differentially expressed proteins with only one predicted membrane protein.

Due to this limited outcome membrane preparations were subjected to a tube-gel trypsin digestion and investigated by nanoflow-liquid-chromatography-coupled tandem mass spectrometry. Based on this approach a total of 212 proteins were detected in MDM including 32 proteins of bovine origin; 275 proteins were detected in CDM; 59% of MDM and CDM proteins were predicted to be membrane-associated. A total of 130 of the proteins were detected in both MDM and CDM and 48 predicted membrane proteins were detected in MDM from at least two cows. Four of these proteins were not detected in CDM, implying differential expression in the host. All membrane-associated proteins, especially the four identified as being differentially expressed, might be relevant targets for further analyses into the pathogenesis of bovine paratuberculosis.