Methods and systems for on-column protein delipidation

• US 7,943,046 B2
• Filed: 06/22/2006
• Issued: 05/17/2011
• Est. Priority Date: 10/01/2004
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Zolotarjova, Nina I., Martosella, James D.
• Primary Examiner(s): Therkorn; Ernest G
• Application Number:
  US11/472,725
• Publication Number:
  US 20060240633A1
• Time in Patent Office: 1,790 Days
• Field of Search: 210/635, 210/656, 210/198.2, 210/502.1, 530/413, 530/417
• US Class Current: 210/635
• CPC Class Codes: C07K 1/16 by chromatography

Abstract

Embodiments of the present invention provide a method of chromatographic delipidation comprising separating a lipid-containing sample on a superficially porous stationary phase at greater than about 70° C., at least about 80° C., having at least one mobile phase comprising an ion-pairing agent in water, an ion-pairing agent in an organic modifier, an acid in an organic modifier, and an alcohol. The invention provides minimal protein losses and high run-to-run reproducibility. The on-column delipidation method aventageously utilize reversed phase liquid chromatography.

Claims

1. An on-column delipidation method comprising separating a lipid-containing sample on a chromatographic column having at least one silica-based stationary phase, at a temperature of at least about 70°

C.;

wherein separation further comprises at least one mobile phase having an alcohol and at least one of the following eluents;

(a) about 0.1 weight % of an ion-pairing agent in water;

(b) about 0.08 weight %, of an ion-pairing agent in organic modifier;

or

(c) about 20 weight % of an acid in an organic modifier, or combinations thereof.

2. The delipidation method of claim 1, wherein at least one of:

eluent (c) is varied from about 0 to about 100 weight % in about 5 minutes; and

the alcohol is varied from about 0 to 100 weight % in about 5 minutes.

3. The method of claim 1, wherein the temperature is greater than about 80°

C.

4. The method of claim 1, wherein the ion-pairing agent comprises at least one of trifluoroacetic acid (TFA), pentafluoroproprionic acid (PFPA), and heptafluorobutyric acid (HFBA).

5. The method of claim 1, wherein the ion-pairing agent comprises at least one of tetramethylammonium chloride, tetrabutylammonium chloride, and triethylamine.

6. The method of claim 1, wherein the organic modifier comprises at least one of acetonitrile, tetrahydrofuran (THF), methylene chloride, ethanol, methanol, ethanol, n-propanol, isopropanol, or combinations thereof.

7. The method of claim 1, wherein the sample is eluted with a gradient comprising increasing amounts of trifluoroacetic acid in at least one organic modifier, and variable concentrations of an acid in an organic modifier, and variable concentrations of an alcohol.

8. The method of claim 7, wherein the organic modifier comprises acetonitrile.

9. The method of claim 7, wherein the alcohol comprises isopropanol.

10. The method of claim 1, wherein the silica-based stationary phase is at least partially superficially porous.

11. The method of claim 10, wherein chromatography is performed by reversed phase-HPLC.

12. The method of claim 11, wherein the eluate is detected upon elution from at least one reversed phase-HPLC column.

13. The method of claim 12, wherein the sample, prior to separation, is solubilized in a solution comprising a strong acid.

14. The method according to claim 13, wherein said acid is formic acid.

15. The method of claim 11, wherein the sample load on the stationary phase comprises from about 100 micrograms to about 2 grams.

16. The method according to claim 11, wherein the superficially porous stationary phase comprises an average particle diameter of about 2 to about 20 micrometers.

17. The method according to claim 11, wherein the reversed phase comprises C₆ to about C₃₀ hydrocarbon selected from the group consisting of alkane, substituted alkane, alkene, substituted alkene, aryl or substituted aryl, and combinations thereof.

18. The method according to claim 11, wherein the reversed phase comprises a silane compound having at least one C₁₀ to about C₃₀ hydrocarbon.

19. The method according to claim 11, wherein the reversed phase comprises at least one divalent silane having a structure:

—

Si(R)(Me)-(CH₂)₃—

Si(R)(Me)-

wherein R is n-octadecyl group, n-tetradecyl group, or mixtures thereof, and Me is methyl.

20. The method according to claim 11, wherein the reversed phase comprises at least one silane of the formula:

A—

O—

SiR₁R₂R₃

where R₁, R₂, and R₃ are each independently alkane, substituted alkane, alkene, substituted alkene, aryl or substituted aryl; and

A is a surface group of the substrate to which the silane is attached.

21. An on-column delipidation method comprising separating a lipid-containing sample on a chromatographic column having at least one superficially porous stationary phase, at a temperature of at least about 70°

C.;

wherein separation further comprises at least one mobile phase having at least one of;

(a) about 0.1 weight % trifluoroacetic acid in water,

(b) about 0.08 weight % trifluoroacetic acid in acetonitrile,

(c) about 20 weight % formic acid in an organic acetonitrile, or

(d) isopropanol,

or combinations thereof.

22. The delipidation method of claim 21, wherein at least one of:

eluent (a) is varied from about 20 to about 50 weight % in about 40 minutes;

eluent (b) is varied from about 50 to about 100 weight % in about 10 minutes;

eluent (c) if varied from about 0 to about 100 weight % in about 5 minutes; and

eluent (d) is varied from about 0 to 100 weight % in about 5 minutes.

23. The method of claim 21, wherein the temperature is greater than about 80°

C.

24. The method of claim 21, wherein the chromatography is performed by reversed phase-HPLC.

25. The method of claim 21, wherein the eluate is detected upon elution from at least one reversed phase-HPLC column.