Protein and peptide purification methods
Provided herein are methods, reagents, and kits for isolating polypeptides, such as a proteome. Also provided herein is a modified trypsin polypeptide that is resistant to autolysis, and that can be selectively-separated from a biological sample once digestion is complete.
1. A coupling compound having the structure:
2. A method of purifying a polypeptide, comprising:
mixing a sample having a basic pH, comprising a polypeptide or a protease-digested polypeptide with an amount of a coupling compound as claimed in claim 1 ;
coupling polypeptides in the sample to a second member of the bio-orthogonal coupling pair linked to a substrate;
optionally washing polypeptides bound to the substrate to remove any unbound materials from the substrate-bound polypeptides; and
eluting the polypeptides from the substrate in an elution solution having an acidic pH.
3. The method of claim 2 , wherein the substrate is a bead, a solid surface or a porous matrix.
4. The method of claim 2 , wherein:
the first member of the bio-orthogonal coupling pair is the tetrazinyl group, and the second member of the bio-orthogonal coupling pair is a strained cycloalkene.
5. The method of claim 2 , wherein the polypeptides of the sample are digested with a modified trypsin polypeptide, comprising:
a trypsin polypeptide;
biotin moieties attached to the trypsin polypeptide; and
groups comprising a member of a bio-orthogonal coupling pair attached to the trypsin polypeptide,
wherein the ratio of groups comprising a member of a bio-orthogonal coupling pair to biotin moieties on the trypsin polypeptide ranges from 1:4 to 1:6.
6. The method of claim 4 , wherein the strained cycloalkene is trans-cyclooctene.