Notes

1. Although it is possible to buy a vacuum manifold specifically for 96-well plates, you can build your own for a fraction of the cost. We make our own out of a tissue culture flask (use a T175-sized flask). Simply cut a portion out of the top of the flask using a hot knife, which is slightly smaller than the size of the 96-well plate. The plate can then sit over this area. Adapt a cap with a piece of tubing that can be attached to a vacuum cap, and seal around the vent of the flask. Place the 96-well plate on top of the flask, and attach the tubing to the vacuum. You now have a very cheap and efficient vacuum drainage system. Be careful not to apply too high a vacuum or the flask will split. The liquid should drain through and collect rapidly in the bottom of the tissue culture flask.

2. 1D and 2D polyacrylamide gel electrophoresis are described elsewhere in this book. However, a good description of 2D electrophoresis that works for most therapeutic proteins is presented in ref. 6. If staining with silver stain before undertaking in-gel digestion, it is imperative that glutaraldehyde is omitted in the sensitizing step.

3. Destaining is usually undertaken until all color has been removed from the gel piece, which may require repetition of this step. However, recent reports suggest that destaining is not required to obtain reproducible and acceptable proteolytic digestion (4).

4. Destaining of silver-stained gels is often easier to undertake using a 1% solution of hydrogen peroxide (H2O2). Destaining should be complete within 5-10 min when using H2O2.

5. The destaining solution should now be colored and the gel piece clear. If not, repeat the destaining step.

6. Incubation can be achieved in a shorter period of time. Incubation for 4 h is usually sufficient to achieve acceptable digestion. Prolonged incubation should be avoided.

7. After overnight incubation at 37°C, most of the liquid will be in the cap of the tube because of evaporation and condensation. It is therefore necessary to spin this down before removing the supernatant.

8. This extraction procedure should extract most peptides out of the gel. It is possible to keep each extraction separate and analyze them separately. Using TFA does suppress ionization during mass spectrometric analysis, particularly when using electrospray mass spectrometry.

9. Peptides can be stored at -80°C for prolonged periods of time. However, for particular samples, it may be difficult to resolubilize peptides if concentrated to dryness. If this is the case, concentrate down to a few microliters, and store in a liquid form.

10. Filling the outside wells with water helps prevent evaporation and drying out of the sample wells during prolonged incubation periods.

11. Aqueous buffers cannot be drawn through the PVDF membrane until treated with methanol.

12. If you have a large volume of protein, this can be applied to the membrane in several steps, drawing the liquid off under vacuum after each application. This is an excellent and quick way of concentrating the protein of interest.

13. This will require passing several lots of reducing buffer through the membrane under vacuum, as 500 |J,L does not fit into the well.

14. It is necessary to block the membrane to prevent the trypsin immobilizing on the membrane once it is introduced.

15. At this stage, N-glycan analysis can be undertaken on the immobilized protein as described in ref. 6.

16. MALDI-TOF mass spectrometry analysis will give a peptide mass fingerprint. However, by using an HPLC connected to an electrospray mass spectrometer, it is possible to obtain both a peptide chromatogram and a mass measurement for each resolved peak. This can improve your chances of observing any changes in your tryptic map owing to differential posttranslational modifications or protein modifications during bioprocessing.

References

1. Smales, C. M., Moore, C. H., and Blackwell, L. F. (1999) Characterization of lysozyme-estrone glucuronide conjugates. The effect of the coupling reagent on the substitution level and sites of acylation. Bioconjug. Chem. 10, 693-700.

2. Smales, C. M., Pepper, D. S., and James, D. C. (2000) Protein modification during antiviral heat bioprocessing. Biotechnol. Bioeng. 67, 177-188.

3. Deutzmann, R. (2004) Structural characterization of proteins and peptides. Methods Mol. Med. 94, 269-297.

4. Terry, D. E., Umstot, E., and Desiderio, D. M. (2004) Optimized sample-processing time and peptide recovery for the mass spectrometric analysis of protein digests. J. Am. Soc. Mass Spectrom. 15, 784-794.

5. Courchesne, P. L., Luethy, R., and Patterson, S. D. (1997) Comparison of in-gel and on-membrane digestion methods at low to sub-pmol level for subsequent peptide and fragment-ion mass analysis using matrix-assisted laser-desorption/ionization mass spectrom-etry. Electrophoresis 18, 369-381.

6. Smales, C. M., Pepper, D. S., and James, D. C. (2002) Protein modification during antiviral heat-treatment bioprocessing of factor VIII concentrates, factor IX concentrates, and model proteins in the presence of sucrose. Biotechnol. Bioeng. 77, 37-48.

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