Peptide purification formulations and methods
Improved formulations for purification of peptide from biological samples and methods and kits for purifying peptides from biological samples (e.g., cells and tissues), as well as use of purified peptides (e.g., polypeptides derived from protein digests) in mass spectrometry (e.g., LC-MS) applications are described.
1 . A method of purifying peptides from a biological sample, comprising:
(a) contacting the biological sample with a hydrophobic, polymer-based cation exchange material under acidic conditions, such that the biological sample binds to the cation exchange material, wherein the biological sample comprises:
(i) at least one peptide,
(ii) at least one monovalent cationic contaminant and/or a at least one hydrophobic contaminant, and
(iii) at least one hydrophilic contaminant;
(b) washing the cation exchange material with a first volatile acidic solution, wherein the first volatile acidic solution comprises 20% or less (v/v) volatile organic solvent, water, and a first volatile acid, such that the at least one peptide is retained on the cation exchange material and the at least one hydrophilic contaminant is removed from the cation exchange material;
(c) washing the cation exchange material with a first volatile salt solution comprising a second volatile acidic solution, and a first volatile base solution, wherein the second volatile acidic solution comprises greater than 20% (v/v) volatile organic solvent, and water,
such that the at least one peptide is retained on the cation exchange material and the at least one monovalent cationic and/or the at least one hydrophobic contaminant is removed from the cation exchange material; and
(d) eluting the retained peptide from the cation exchange material with a volatile alkaline or neutral solution, wherein the volatile alkaline or neutral solution comprises a volatile organic solvent, water, and a volatile compound selected from a first or a second volatile base, a first or a second volatile salt, and a combination thereof.
2 . The method of claim 1 , wherein the hydrophilic contaminant is a neutral contaminant, an anionic contaminant, or a combination thereof.
3 . The method of claim 1 , wherein the biological sample further comprises a salt, detergent, a lipid, a small neutral molecule, or a combination thereof.
4 . The method of claim 1 , comprising contacting the sample with the hydrophobic, polymer-based cation exchange material at a pH 1-5.
5 . The method of claim 1 , comprising washing the cation exchange material at a pH 1-5.
6 . The method of claim 1 , comprising eluting the sample from the cation exchange material at greater than pH 5.
7 . The method of claim 1 , wherein the monovalent cationic contaminant is selected from the group consisting of ammonium, sodium, potassium, tris(hydroxymethyl)aminomethane (tris), hydroxylamine, ethanolamine, HEPES (4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid), PIPES (piperazine-N,N′-bis(2-ethanesulfonic acid)), EPPS (4-(2-Hydroxyethyl)-1-piperazinepropanesulfonic acid), glycine, a mass tag, and a combination thereof.
8 . The method of claim 1 , wherein the monovalent cationic contaminant is or comprises a mass tag or derivative thereof.
9 . The method of claim 1 , wherein hydrophobic contaminant is a lipid or a detergent.
10 . The method of claim 1 , wherein the hydrophilic contaminant is a salt, nucleic acid, or a carbohydrate.
11 . The method of claim 1 , further comprising treating the biological sample with a proteolytic enzyme to produce the peptide prior to contacting the biological sample with the cation exchange material.
12 . The method of claim 1 , wherein the hydrophobic, polymer-based cation exchange material is sulfonated divinyl benzene polystyrene, sulfonated polydivinyl benzene, or sulfonated divinyl benzene/polystyrene/pyrrolidone resin.
13 . The method of claim 1 , wherein the first and/or second volatile acid is selected from the group consisting of formic acid, acetic acid, trifluoracetic acid, trichloroacetic acid.
14 . The method of claim 1 , wherein the first and/or second volatile base is selected from the group consisting of trimethylamine, ammonia, triethylamine, piperidine, and butylamine.
15 . The method of claim 1 , wherein the volatile organic solvent is selected from the group consisting of acetonitrile, methanol, ethanol, n-propanol, iso-propanol, acetone, and a combination thereof.
16 . The method of claim 1 , wherein the sample comprises two or more peptides, a digested protein, or a polypeptide, a labeled peptide, a labeled peptide digest, a mass tag labeled peptide, a mass tag labeled peptide digest, a tandem mass tag (TMT)-labeled peptide, or a tandem mass tag (TMT)-labeled peptide digest.
17 . The method of claim 11 , wherein the proteolytic enzyme is trypsin, Lys-C, AspN, or GluC.
18 . The method of claim 1 , wherein the first and/or second volatile salt is selected from ammonium acetate, ammonium formate, ammonium trifloroacetate, triethylammonium formate, triethylammonium acetate, triethylammonium trifluoroacetate and ammonium bicarbonate.