PFG Digestion protocol for in-solution digests

A key step in almost any proteome analysis is the conversion of protein to (usually tryptic) peptides. This step is so critical because a) it generates analytes that are appropriately charged for MS analysis, and b) are compatible with the nanoscale chromatography systems we use. Failure to achieve complete digestion can lead to missed cleavage (which weakens the strength of database searches) and worse, led to (pre)column clogging.

We should therefore adopt a common digestion protocol that will work for most analyses. The aim is to load onto the column an appropriate amount of material such that a typical protein will be represented at approx 50fmol. In order to calculate this load, it is necessary to make some assumptions. First, that the recovery of material after all digestion/preparation protocols is 100%, secondly, the molecular weight of an ‘average’ protein for complex protein mixtures, and c) the overall complexity of the mixture.

We can assume that a typical protein has a molecular weight of 40kDa. Thus: 40,000g = 1 mol, 40g = 1mmol, 40mg=1umol, 40µg=1nmol, 40ng=1pmol, 40pg=1fmol. Thus, 2ng=50fmol, the amount loaded on column.

This only works for pure proteins. For a mixture, we need to factor in complexity (C). Let’s assume that all proteins are equimolar for now. If the protein is pure, the complexity (C) is 1, such that 2ng loaded should give a good LC-MS/MS trace. If C=10, then if you load 2ng, you’ll only get 5fmol of each protein. Therefore:

• If C=10, you need to load a digest equivalent of 20ng
• If C=100, you need to load 200ng
• If C=250, you need to load 500ng
• If C=1000 you need to load 2µg
• For most samples, C>1000, but actually, the dynamic range means that you can assume C=1000

Digestion protocol
This protocol is adopted from the Waters recommendations. The features are the use of a digestion enhancer (Rapigest) that is destroyed after digestion, b) an obligatory reduction and alkylation – this should henceforth be performed as a routine. The digestion is based on large volumes (200uL digest) and large amounts of material (100ug), and can be scaled downwards within reason, say to one quarter of these volumes (a 50uL digest).