5.3. Conventional proteomics using two-dimensional gel electrophoresis

Genomic sequences are not sufficient for explaining biological functions because there is no strict linear correlation between the genome and the proteome of an organism. For example, protein modifications and relative concentration of proteins cannot be determined by genomic analysis (Pandey and Mann, 2000). Furthermore, the DNA sequences give no information about conditions and time for translation as well as effects of up or down regulation of gene expression (Humphery-Smith et al., 1997). Therefore, predictions on the basis of genetic information should be completed by expression data at the level of the transcriptome and the proteome. By means of proteome analysis, a holistic approach of protein expression under specific conditions is possible. The classical method of proteomics is the two-dimensional (2D) gel electrophoresis (O’Farrell, 1975; Klose, 1975). Recently, it was successfully used for a comparison of the P. larvae genotypes ERIC I and II (Fünfhaus and Genersch, 2012).

    Sample preparation for 2D electrophoresis uses the following protocol (Fünfhaus and Genersch, 2012):

  1. Cultivate P. larvae strains to be analysed on Columbia sheep blood agar (CSA; see section 3.1. for recipe) plates for three days at 37°C.
  2. To obtain a pre-culture, inoculate 3 ml brain heart infusion broth (BHI, see section 3.1 for recipe) with one bacterial colony and the cells are grown overnight at 37°C with shaking at 200 x g.
  3. To obtain a 10 ml main culture, inoculate 9 ml BHI with a pre-culture to achieve a final OD600 of 0.01 after adjustment to a final volume of 10 ml with BHI.
  4. Incubate at 37°C with shaking at 200 x g.
  5. Monitor growth continuously by measuring OD600.
  6. Stop growth in the late exponential phase (OD600 0.65) by harvesting the cells via centrifugation (20 min, 5,000 x g, 4°C).
  7. Wash the bacterial cell pellets three times with ice-cold PBS.
  8. Resuspended in 1 ml lysis buffer (7 M urea, 2M thiourea, 4 % (w/v) CHAPS, complete protease inhibitor cocktail (Roche)).
  9. Disrupt  cells by using a sonicator, e.g. ranson Sonifier 250 (duty control: 10 %; output control: 1). Repeated sonication cycles (ten times for 30 sec) are interrupted by cooling phases for 60 sec.
  10. Incubate samples for 1 h at RT to facilitate dissolving the proteins.
  11. Separate crude protein extracts from cellular debris by centrifugation at 16,100 x g for 25 min at 4°C.
    The resulting supernatant contains the cytosolic proteins but also salts and other small charged molecules which need to be removed from the solution by precipitation.
  12. Precipitate the cytosolic proteins of P. larvae with one volume 20 % TCA, 2 % Triton X-100 overnight at 4°C.
  13. Pellet the precipitated proteins by centrifugation (16,100 x g, 25 min, 4°C).
  14. Wash the pellets with 80 % acetone to remove residual TCA.
  15. Resuspend the washed pellet in 200 µl sample buffer (7 M urea, 2M thiourea, 4% (w/v) CHAPS, 100 mM DTT, 1 % Bio-Lyte Ampholyte (Bio-Rad), complete protease inhibitor cocktail (Roche)).
  16. Vortex for 1 h at RT.
  17. Separate the insoluble material from the soluble proteins by centrifugation (15,000 x g, 1 h, 15°C) to obtain the soluble cytoplasmic fraction.
  18. Determine the protein concentration by performing the Pierce® 660 nm Protein Assay (Thermo Scientific) according to the manufacturer’s protocol.
  19. Store samples at -80°C until further analysis.
            These protein samples are then subjected to 2D-gel electrophoresis with the isoelectric focussing (IEF) as the first dimension followed by SDS-PAGE analysis as second dimension.
  20. Dilute the samples with rehydration buffer (7 M urea, 2 M thiourea, 1 % (w/v) CHAPS, 10 mM DTT, 0.25 % Bio-Lyte Ampholyte (Bio-Rad)).
  21. Determine the amount of protein to load on IPG strip according to the sample, the pH range, the length of the IPG strip and the staining method.
  22. Focussing of proteins is best performed in commercially available, immobilized pH gradient strips (IPG strips) selecting a suitable pH gradient. For the analysis of P. larvae proteins IPG strips with a pH gradient 5-8 and a length of 7 cm (Bio-Rad) proved to be useful.
  23. Load 60 µg cytosolic P. larvae proteins in a total rehydration volume of 125 µl on the IPG strips.

            The following protocol for IEF is adapted to the PROTEAN IEF Cell (Bio-Rad) at 20°C:
  24. Load the samples on the IPG strips by an active in-gel rehydration for 18 h at 50 V followed by a voltage profile with increasing values:
    • linear increase from 50 – 200 V for 1 min.
    • 200 V for 200 Vh.
    • linear increase from 200 – 500 V for 1 min.
    • 500 V for 500 Vh.
    • linear increase from 500 – 1000 V for 1 min.
    • 1000 V for 1000 Vh.
    • linear increase from 1000 – 2000 V for 1 min.
    • 2000 V for 2000 Vh.
    • linear increase from 2000 – 4000 V for 1 min.
    • 4000 V for 4500 Vh.
  25. Subsequently, saturate the proteins separated in the IEF gel with SDS by equilibrating the IPG strips in equilibration buffer I (6M urea, 30 % (v/v) glycerol, 5 % (w/v) SDS, 0.05 M Tris pH 8.8, 1 % (w/v) DTT) for 10 min.
  26. Block free SH-groups of the separated proteins by equilibrating the IPG strips in equilibration buffer II (6M urea, 30 % (v/v) glycerol, 5 % (w/v) SDS, 0.05 M Tris pH 8.8, 5 % (w/v) iodoacetamide) for 10 min.
  27. For the SDS-PAGE as second dimension a 12 % polyacrylamide gel run at 35 mA in a PROTEAN II XL Cell (Bio-Rad) proved to be suitable.
  28. Gels are stained with Coomassie (Page Blue Protein Solution, Fermentas) according to standard protocol (see the BEEBOOK paper on physiological and biochemical methods (Hartfelder et al., 2013)).

         Analysis of the 2D gels can be performed by using software PDQuest 8.0 (Bio-Rad). Protein identification can be achieved by mass spectrometric analysis followed by comparison of peptide masses and sequence information of the sample with different databases (see the BEEBOOK paper on physiological and biochemical methods (Hartfelder et al., 2013) and the BEEBOOK paper on chemical ecology (Torto et al., 2013)).