Careful and consistent sample preparation is the first step to quality peptide mapping data.
Protein digestion for peptide mapping is among the most complicated sample prep to be done in when monitoring critical quality attributes – perhaps rivaled only by sample prep for glycan analysis. Agilent’s Peptide Mapping “How to” Guide lays out a general protocol, but there are always proteins or matrices that need special consideration. There are multiple steps, each with choices to be made and opportunity for optimization. Putting some thought into your sample prep procedure can make a big difference in the separation and detection of your peptides. I’ve quizzed a few colleagues (thanks, Suresh, Veronica, and Rita!), and based on our collective experience we’ve come up with these tips.
Some matrices require clean up or adjustments for more efficient digestion. An affinity capture step can be used to either deplete matrix proteins such as albumin and immunoglobulins or to enrich the target protein based on PTMs such as phosphorylation or glycosylation.
- Think about your whole workflow early on – if you’re planning to use MS for detection, think carefully about the reagents you use and how much you use. How easy are they to remove from the sample in a clean up step? Will they lead to ion suppression?
- Consider taking a blank sample through your entire process as a control for troubleshooting purposes (sample prep through any enrichment and clean up).
- pH can be a balancing act – lower pH can help prevent deamidation, but many proteases work more efficiently at slightly elevated pH. (Don’t you hate choosing the lesser of two evils?)
Denaturation, Reduction, and Alkylation
For the protease to efficiently access cleavage sites, its necessary to denature the protein and reduce and alkylate the disulfide bonds (unless of course you’re looking to map the disulfide bonds).
- If you’re using urea to denature, don’t heat the sample! This will lead to lysine carbamylation, and we don’t (usually) want to add any PTMs.
- Guanidine can inhibit protease activity, so be sure to dilute or remove it prior to digestion.
- Highly concentrated or complex proteins can be prone to precipitation during the denaturation step, so keep an eye out. Dilution before denaturation may help.
- DTT and mercaptoethanol are really stinky reductants. TCEP is just as effective as DTT, and it’s odorless (a good enough reason in my book!). Just beware it’s not particularly stable in phosphate buffer at neutral pH, so a fresh solution is necessary.
Trypsin is the most commonly used protease since it’s cleavages are so well defined and predictable, but there are a variety of digestion agents available if trypsin’s lysine and arginine cleavages don’t produce suitable peptides for separation and detection. We’ll talk more about this in a future post. The optimum pH is dependent on the chosen digestion reagent, but digestion time, temperature, and protease concentration are variables that are independent on one another as well as the target protein and protease.
- Trypsin cleavage is slower when Lys or Arg are adjacent to Cys, Glu, or Asp, and doesn’t occur at all when Lyr or Arg is followed by Pro.1
- Two separate additions of trypsin may help achieve complete digestion.
- After digestion, acidify the sample with 0.1% formic acid rather than 1%.
- Many protocols proscribe a certain incubation or reaction time, but if you’re implementing a protocol to use long-term, it would be worth an experiment to see how long that step really takes. Many a grad student (yours truly included) has let a reaction go for 12 hours because they wanted to go home for the night!
- Trypsin (or other protease) autolysis can lead to extra peaks in your peptide map. Some trypsin products are more prone to this than others. Just something to keep in the back of your mind if you’re troubleshooting coelution or chasing unidentified peaks (something your blank control can help with).
Trypsin is so prevalent that protocols are plentiful, but while we’re on the topic, I wanted to mention this Nature Protocols article by Giansanti et al that details digestion protocols for six other proteases along with discussion of the pros and cons of each.1
Enrichment and Clean Up
If you’re looking for specific PTMs such as phosphorylation or glycosylation, enrichment following digestion may be helpful. Clean up is often necessary for successful analysis of your peptide map, especially with MS detection as salts can significantly limit ionization and therefore sensitivity.
- Desalt the sample before MS analysis for best sensitivity. Salt can significantly limit how well the sample ionizes.
- If the sample appears opaque or cloudy, filter it before injection.
A final general tip – once you have settled on a protocol, stick to it! Any variation from the protocol can lead to inconsistent results and more questions.
Talk soon –
- Giansanti, L. Tsiatsiani, T.Y. Low, A.J.R. Heck. Six alternative proteases for mass spectrometry-based proteomics beyond trypsin. Nature Protocols, 2016.
Keywords: Bio columns, liquid chromatography, tips and tricks, peptide mapping, sample prep, AdvanceBio blog