First, let’s define ‘large’. The Proteome Discoverer sets the upper limit for a precursor ion at 10,000 Da. This means anything bigger than 10 kDa will not be considered even if it’s present in the MS data, and a different software package ($$) will be required to analyze the high MW data. Clearly, proteolytic peptides with MW > 10 kDa will not be very useful for protein identification. I suggest using a different enzyme or a combination of enzymes. I have seen tryptic peptides up to 7 kDa in some in-gel digested samples, so apparently some large peptides do come out of gel.
Next, a large peptide’s physico-chemical properties (e.g. hydrophobicity, pI, hydrodynamic radius) must be considered as they will affect the extraction efficiency. If the peptide’s properties are known, the extraction solvent composition and pH can be adjusted to improve the peptide’s solubility.
Finally, let’s consider the gel from which the large peptides need to come out. Obviously, it will be easier to get the large peptides out of a 4 %T gel than out of a 20 %T one. Soaking a gel piece in deionized water and then freezing it should crash enough pores in the gel to improve the extraction of large peptides (water expands as it freezes). Additionally, the gel could be ‘squeezed out’ a few times by changing extraction solvent from neat acetonitrile to an aqueous mixture. The gel piece will shrink in acetonitrile expelling the peptide solution. Re-hydrating the gel and then shrinking it again in acetonitrile will ‘squeeze out’ more of the digest.
Using elevated temperature (50 C), vortex mixer, and/or ultrasonic bath should all improve the extraction. Use common sense: 50C and a high pH buffer is not a good idea for the phosphopeptide extraction. Another word of caution: don’t get carried away. Three extraction steps should be enough. If you end up with a large volume (e.g. more than 0.5 mL), the benefits of a thorough extraction might become negated by the losses due to dilution. Peptides and proteins tend to adhere to the polypropylene tubes. A large volume of a dilute peptide solution presents a large surface area for the peptides to adsorb.
What to do if this doesn’t work? You can try in-solution digestion. If the mixture is too complex and a PAGE step is necessary, you can try electroeluting the protein(s). Intact proteins electroeluted from gel bands can be buffer-exchanged using small-volume 3,000 Da MWCO spin columns and proteolyzed in solution.
