Method for separating peptides and proteins
View Patent ↗Embodiments of the present invention are directed to articles of manufacture, devices, methods and apparatus for performing liquid chromatography featuring a chromatographic sorbent having one or more pentafluorophenyl groups, wherein said one or more pentafluorophenyl groups are a bonded phase on a sorbent selected from the group comprising silica, organic polymers or hybrid organic silane material and said pentafluorophenyl groups are in a mono-, bi-, and tridentate forms.
1. A chromatographic method comprising the steps of:
loading a sample potentially comprising an analyte onto a mixed-mode chromatographic sorbent, wherein the analyte comprises a modified peptide or a modified protein and wherein
the sorbent conforms to Formula 1:
(W 1 ) a (W 2 ) b (W 3 ) c Si(Z—C 6 F n H 5-n ) d Formula 1
where W 1 , W 2 , and W 3 are selected from the groups consisting of hydrogen, hydroxyl, hydroxy-aliphatic, aliphatic, oxygen, nitrogen, and silane wherein at least one of W 1 , W 2 , and W 3 separately represent terminal silane valences bonded to oxygen, nitrogen, carbon or silane atoms of a support, Z represents a aliphatic moiety and n is an integer from 1-5, and a+b+c+d=4; where a is 1-3;b+c is less than or equal to 2, and d is 1-3;
eluting the mixed-mode chromatographic sorbent in reverse phase mode, thereby separating the analyte, if present, from the sample; and
eluting the mixed-mode chromatographic sorbent in ion exchange mode, thereby regenerating the mixed-mode chromatographic sorbent.
2. The method of claim 1 , further comprising analyzing the analyte by LC/MS.
3. The method of claim 2 , wherein the analyte eluent is substantially free of salt ions, thereby facilitating MS analysis without salt ion interference or first requiring the removal of salt ions.
4. The method of claim 1 , wherein the ion exchange is cation exchange.
5. The method of claim 1 , wherein the ion exchange is anion exchange.
6. The method of claim 1 , wherein the analyte consists essentially of one or more phosphorylated peptides or phosphorylated proteins.
7. The method of claim 1 , wherein the analyte consists essentially of one or more charged glycopeptides or glycoproteins.
8. The method of claim 1 , wherein the analyte consists essentially of one or more sialylated glycopeptides or glycoproteins.
9. The method of claim 1 , wherein the chromatographic sorbent comprises one or more pentafluorophenyl groups.
10. The method of claim 1 , wherein the sorbent comprises a silica, organic polymer, or hybrid organic silane material and one or more pentafluorophenyl groups bonded thereto.
11. The method of claim 1 , wherein Z is —(CH 2 ) m — where m is 2 or 3.
12. The method of claim 1 , wherein the support is silica where the terminal silane valences are bonded to silane.
13. The method of claim 1 , wherein n is 5.
14. The method of claim 1 , wherein said surface represents a distribution of the structure of Formula 1 with a distribution of monodentate, bidendate, and tridentate forms.
15. The method of claim 14 , wherein the distribution comprises 1-30% monodentate form, 50-90% bidentate form, and 1-50% tridentate form.
16. The method of claim 14 , wherein the distribution comprises 2-25% monodentate form, 60-80% bidentate form, and 10-40% tridentate form.
17. The method of claim 1 , wherein the modified peptide or modified protein comprises a phosphorylated peptide, glycopeptide, charged glycopeptide, lapidated peptide, prenylated peptide, sulfated peptide, hydroxylated peptide, acetylated peptide, phosphorylated protein, glycoprotein, charged glyco protein, lapidated protein, prenylated protein, sulfated protein, hydroxylated protein, acetylated protein, or a combination thereof.
18. A chromatographic method comprising the steps of:
loading a sample potentially comprising an analyte onto a mixed-mode chromatographic sorbent, wherein the analyte comprises at least one modified peptide or modified protein and wherein the sorbent conforms to Formula 1:
(W 1 ) a (W 2 ) b (W 3 ) c Si(Z—C 6 F n H 5-n ) d Formula 1
where W 1 , W 2 and W 3 are selected from the groups consisting of hydrogen, hydroxyl, hydroxy-aliphatic, aliphatic, oxygen, nitrogen, and silane wherein at least one of, W 1 , W 2 , and W 3 separately represent terminal silane valences bonded to oxygen, nitrogen, carbon or silane atoms of a support, Z represents a aliphatic moiety and n is an integer from 1-5 and a+b+c+d=4; where a is 1-3; b+c is less than or equal to 2, and d is 1-3;
eluting the mixed-mode chromatographic sorbent in reverse phase mode, thereby separating the analyte, if present, from the sample; and
eluting the mixed-mode chromatographic sorbent in cation exchange mode, thereby regenerating the mixed-mode chromatographic sorbent.
19. The method of claim 17 , further comprising analyzing the analyte by LC/MS.
20. The method of claim 18 , wherein the analyte eluent is substantially free of salt ions, thereby facilitating MS analysis without salt ion interference or first requiring the removal of salt ions.
21. The method of claim 18 , wherein the modified peptide or modified protein comprises a phosphorylated peptide, glycopeptide, charged glycopeptide, lapidated peptide, prenylated peptide, sulfated peptide, hydroxylated peptide, acetylated peptide, phosphorylated protein, glycoprotein, charged glyco protein, lapidated protein, prenylated protein, sulfated protein, hydroxylated protein, acetylated protein, or a combination thereof.