Hydrophobicity shifted enzyme
The present invention relates to a method for analyzing a sample, the method comprising: (a) incubating a sample comprising an analyte with at least one enzyme to produce a digestion mixture comprising fragments of the analyte; (b) loading the digestion mixture onto a reversed-phase chromatography column; and (c) performing reversed-phase chromatography on the digestion mixture, wherein the at least one enzyme is hydrophobically modified to increase a retention time of the at least one enzyme such that the at least one enzyme elutes from the reversed-phase chromatography column later than the fragments of the analyte.
1 . A method for analyzing a sample, the method comprising:
(a) incubating a sample comprising an analyte with hydrophobically modified trypsin to produce a digestion mixture comprising fragments of the analyte;
(b) loading the digestion mixture onto a reversed-phase chromatography column; and
(c) performing reversed-phase chromatography on the digestion mixture, wherein the hydrophobically modified trypsin is hydrophobically modified at the C-terminus with a sequence extension comprising at least three hydrophobic amino acid residues to increase a retention time of the hydrophobically modified trypsin such that the hydrophobically modified trypsin elutes from the reversed-phase chromatography column later than the fragments of the analyte.
2 . The method of claim 1 , wherein the hydrophobically modified trypsin is a hydrophobically modified porcine trypsin.
3 . The method of claim 1 , wherein the hydrophobically modified trypsin is resistant to autolysis.
4 . The method of claim 1 , wherein the hydrophobically modified trypsin is derivatized by alkylation of lysine residues of trypsin.
5 . The method of claim 1 , and wherein a stoichiometric ratio of the hydrophobically modified trypsin to the analyte in step (a) is from 1:20 to 2:1, and wherein step (a) comprises incubating the sample with the hydrophobically modified trypsin: at a pH of from 5 to 9; at a temperature of from 20° C. up to a melting temperature of the hydrophobically modified trypsin, and for a time period of less than or equal to 4 hours.
6 . The method of claim 5 , wherein the stoichiometric ratio of the hydrophobically modified trypsin to the analyte in step (a) is 1:1.
7 . The method of claim 1 , wherein a stoichiometric ratio of the hydrophobically modified trypsin to the analyte in step (a) is from 1:20 to 2:1.
8 . The method of claim 1 , wherein the hydrophobically modified trypsin is part of a fusion protein, wherein the fusion protein further comprises a domain or partial domain from a biologically derived protein domain, and wherein the domain or partial domain has a hydrophobicity index greater than 25.