What is your aim with the assay? What do you want to do with the peptides? I take it that this is for mass spectrometric analysis? You should empirically test for a suitable amount of pepsin (usually in the range of 1:100 or so molar ratio to your protein). The specificity of pepsin depends on the pH - I guess at pH 2 pepsin digestion is quite reproducible. Also, temperature and solution content (like ionic strength) plays a role.

I used to do HDX assays with pepsin digestion and picked several time points of my digestion reaction, mixed with alpha-Cyano-4-hydroxycinnamic acid, transferred on an anchor plate and allowed to dry, which of course stopped the proteolysis too. After this, MALDI-TOF analysis, which proved to a relatively easy way of mapping the protein, especially with the help of MS/MS. I also tried to use LC-MS to separate the peptides but this had its own (instrumental) problems mainly considering the HDX.

Later on I switched the pepsin to trypsin and did all of this for my folded and unfolded protein as well as with an interaction partner - a typical old-school protection assay.

EDIT: Oh yeah, forgot to mention. The concentration of proteins in solution can naturally be quite low - in the nanomolar regime - as MS is a very sensitive technique. Hope this helps.

Thank you Arne, I have to do exactly the same!

I would like to do HDX assay with pepsin digestion picking various time points to study the refolding pathway of this domain. The picked points will be analyzed by MALDI-TOF. My concerns were about the pepsin reproducibility: so at pH=2 and with a molar ratio of 1:100 it is reproducible? In your experience, at which temperature do I perform the assay? A tipical Ambic buffer could be used?

porcine and bovine pepsin works at pH between 2 and 5, above that it quickly inactivates. you can stop digestion by increasin pH to 8. it digest quicker at 37 C, but can eat nearly everything, so digestion at lower temperature is easier controled.

You should make test digestions to obtain an optimal amount of pepsin for your needs, as it might actually digest very rapidly, especially if your protein is unfolded or contains loops etc. At pH 2 most proteins are readily unfolded. Try molar ratios like 1:50, 1:100, 1:200, 1:500 etc. and pick different timepoints to get an idea on the rate of the proteolysis.

By increasing the pH, the specificity (or should I say selectivity) of pepsin may change. To my understanding it has a preference over aromatic and acidic residues at pH 2, although pepsin can cleave between pretty much any two amino acids. At pH 2 the reproducibility should be somewhat good despite of this.

The reproducibility can be also increased by using an on-line system with a column with immobilized pepsin. Keeping the flow rate (= the interaction time between your protein and the pepsin) constant, the reproducibility is better. Also, you will reduce the background peptides coming from pepsin itself, as it cannot cleave itself in the column. The on-line system works especially well in HDX experiments, but be sure to lower the temperature (0 ºC) to really quench the hydrogen-deuterium exchange after incubation.

Remember that you can also control the rate of proteolysis by varying the ionic strength and lowering the temperature. If your protein needs salt, you can try ammonium bicarbonate or phosphate, as they are also much more compatible with mass spectrometry than, say, sodium chloride. Also, as Pal pointed out, pepsin can be inactivated, but the pH should be increased rapidly. It doesn't, however, work in HDX if you wish to quench the exchange.

Good luck with your experiments!

Thank a lot Arne, your information are very useful! I'll let you know about the experiment.

:-)

Hi Pal, thanks for your answer, but I need to keep the pH at acidic values due to the HDX experiment.

once pepsin is inactivated by high pH, it does not activates again when lowering the pH subsequently.

Pal, that is true but increasing the pH over 8 will disturb hydrogen-deuterium exchange, which at near-neutral pH can occur on a sub-second time-scale. HDX experiments are very sensitive to pH.

The idea behind these experiments is to incubate a protein in deuterium oxide and solvent exposed protons will exchange to deuterium. After this the reaction will be quenched by lowering the pH and adding pepsin, which digests the protein to peptides and allows the identification of solvent-exposed surfaces in proteins with the help of mass spectrometry. If you inactivate the pepsin by increasing the pH, the exchange reaction on the generated peptides will not be quenched anymore, allowing further exchange (and back-exchange) to occur on the peptides and the information regarding the folded protein is lost.