# US 11,111,268 B2 · Use of quaternary and tertiary ammonium cations to denature proteins

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- Number: US 11,111,268 B2
- Document ID: US11111268B2
- Status: Granted
- Kind: B2
- Application number: 16/323,247
- Filed: 2017-08-17
- Published: 2019-06-06
- Granted: 2021-09-07
- Earliest claimed: 2016-08-17
- Assignees: Agilent Technologies, Inc.
- Applicants: ProZyme, Inc.
- Inventors: Michael J. Kimzey; Francis T. Haxo; Vaishali Sharma
- Web page: https://patentgrep.com/patent/US11111268B2

## CPC

- C07K 1/1136: {by reversible modification of the secondary, tertiary or quarternary structure, e.g. using denaturating or stabilising agents}
- C07K 1/113: without change of the primary structure
- C11D 1/62: Quaternary ammonium compounds
- C11D 17/00: Detergent materials or soaps characterised by their shape or physical properties (shaping soap C11D13/14)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C11D: DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL

## Abstract

Ammonium cation detergents comprising a quaternary or tertiary ammonium cation can be used as detergents to denature proteins and are particularly useful in denaturing glycoproteins or glycopeptides prior to enzymatic deglycosylation. Ammonium cation detergents with sulfate or sulfonate anions are particularly useful.

## Claims

### Claim 1

A method of denaturing a glycoprotein or glycopeptide of interest in vitro, said method comprising incubating said glycoprotein or glycopeptide in vitro with a solution comprising an effective amount of an ammonium cation sulfate or sulfonate detergent comprising (a) an aliphatic chain of 8-24 carbons, (b) a sulfate or sulfonate anion and (c) a tertiary or quaternary ammonium cation, wherein said aliphatic chain is covalently attached to said anion, for a time T to denature said glycoprotein or glycopeptide, thereby denaturing said glycoprotein or glycopeptide.

### Claim 2 (depends on 1)

The method of claim 1, wherein said anion is a sulfate anion.

### Claim 3 (depends on 1)

The method of claim 1, wherein said anion is a sulfonate anion.

### Claim 4 (depends on 1)

The method of claim 1, wherein said chain of 8-24 carbons is saturated.

### Claim 5 (depends on 1)

The method of claim 1, wherein said aliphatic chain is of 8-15 carbons.

### Claim 6 (depends on 1)

The method of claim 1, wherein said aliphatic chain is of 8-13 carbons.

### Claim 7 (depends on 1)

The method of claim 1, wherein said aliphatic chain is 12 carbons.

### Claim 8 (depends on 7)

The method of claim 7, wherein said aliphatic chain is saturated.

### Claim 9 (depends on 1)

The method of claim 1, wherein said aliphatic chain is in a ring configuration.

### Claim 10 (depends on 1)

The method of claim 1, wherein said sulfate or sulfonate anion is covalently attached to said aliphatic chain by being attached to a benzyl which is attached to said aliphatic chain.

### Claim 11 (depends on 1)

The method of claim 1, wherein said cation is a quaternary ammonium cation.

### Claim 12 (depends on 11)

The method of claim 11, wherein said quaternary ammonium cation is selected from the group consisting of:

### Claim 13 (depends on 12)

The method of claim 12, wherein said quaternary ammonium cation is a tetramethylammonium cation.

### Claim 14 (depends on 12)

The method of claim 12, wherein said quaternary ammonium cation is a tetrabutyl, a tetraethyl, or a tetrapropyl ammonium cation.

### Claim 15 (depends on 1)

The method of claim 1, wherein said ammonium cation detergent is tetra methyl dodecyl ammonium sulfate.

### Claim 16 (depends on 1)

The method of claim 1, wherein said cation is a tertiary ammonium cation.

### Claim 17 (depends on 16)

The method of claim 16, wherein said tertiary ammonium cation is a trimethyl, tributyl, triethyl or tripropyl ammonium cation.

### Claim 18 (depends on 1)

The method of claim 1, wherein said glycoprotein or glycopeptide of interest is in a mixture of glycoproteins or glycopeptides.

### Claim 19 (depends on 1)

The method of claim 1, wherein said glycoprotein or glycopeptide of interest is in a cell lysate, blood serum, blood plasma, is a fusion protein, or is a cell membrane protein.

### Claim 20 (depends on 1)

The method of claim 1, further comprising the steps of heating a solution comprising said glycoprotein or glycopeptide and said ammonium cation sulfate detergent or said ammonium cation sulfonate detergent to a temperature ranging from about 80° to about 120° C., maintaining said mixture within said temperature range for time T, and then cooling said solution.

### Claim 21 (depends on 20)

The method of claim 20, wherein said temperature range is from about 90° to about 100° C.

### Claim 22 (depends on 20)

The method of claim 20, wherein said solution is cooled to a temperature of about 35-60° C. following said time T.

### Claim 23 (depends on 22)

The method of claim 22, wherein said solution is cooled to a temperature of about 50° C.

### Claim 24 (depends on 20)

The method of claim 20, further wherein said time T is between about 1 to about 10 minutes.

### Claim 25 (depends on 24)

The method of claim 24, further wherein said time T is about 3 minutes.

### Claim 26 (depends on 1)

The method of claim 1, further comprising releasing glycans from said denatured glycoprotein or glycopeptide by incubating said denatured glycoprotein or glycopeptide with a deglycosylation enzyme for a time sufficient to release said glycans, thereby forming a solution comprising said glycans, said glycoprotein or glycopeptide from which said glycans have been released, and said ammonium cation sulfate or sulfonate detergent.

### Claim 27 (depends on 26)

The method of claim 26, further wherein either said glycoprotein or glycopeptide or said deglycosylation enzyme is immobilized on a solid support.

### Claim 28 (depends on 27)

The method of claim 27, wherein said denatured glycoprotein or glycopeptide is immobilized on a solid support prior to being contacted with said deglycosylation enzyme.

### Claim 29 (depends on 26)

The method of claim 26, further wherein said deglycosylation enzyme is an amidase.

### Claim 30 (depends on 29)

The method of claim 29, further wherein said amidase is PNG F.

### Claim 31 (depends on 26)

The method of claim 26, further wherein said released glycans are labeled following release from said glycoprotein or glycopeptide.

### Claim 32 (depends on 31)

The method of claim 31, further wherein said released glycans are released as β-glycosylamines.

### Claim 33 (depends on 31)

The method of claim 31, further wherein said label is fluorescent.

### Claim 34 (depends on 31)

The method of claim 31, further wherein said labeled released glycans are analyzed.

### Claim 35 (depends on 34)

The method of claim 34, further wherein said analysis is selected from the group consisting of high-performance liquid chromatography, hydrophilic interaction chromatography, nuclear magnetic resonance, fluorescence analysis, Western blotting, gel electrophoresis, capillary electrophoresis, microfluidic separation, and mass spectrometry or a combination of any two or more of these.

### Claim 36 (depends on 34)

The method of claim 34, wherein said analyzing is by detecting a fluorescent signal from said labeled released glycans.

### Claim 37 (depends on 26)

The method of claim 26, further comprising analyzing said denatured glycoprotein (now deglycosylated) or glycopeptide from which said glycans have been released.

### Claim 38 (depends on 37)

The method of claim 37, further wherein said analyzing of said glycoprotein or glycopeptide from which glycans have been released is by high-performance liquid chromatography, hydrophilic interaction chromatography, nuclear magnetic resonance, Western blotting, gel electrophoresis, fluorescence analysis, capillary electrophoresis, microfluidic separation, mass spectrometry, or a combination of two or more of any of these.

### Claim 39 (depends on 37)

The method of claim 37, wherein said analysis is by mass spectrometry.

### Claim 40 (depends on 1)

The method of claim 1, further wherein said solution comprises a reductant, an alkylant, an additional organic solvent denaturant, a chaotrope, or a combination of any of these.

### Claim 41 (depends on 34)

The method of claim 34, further wherein said ammonium cation sulfate detergent or said ammonium cation sulfonate detergent is removed prior to said analyzing.

### Claim 42 (depends on 41)

The method of claim 41, wherein said removing of said ammonium cation sulfate detergent or said ammonium cation sulfonate detergent comprises flowing said solution comprising said released glycans, said glycoprotein or said glycopeptide from which said glycans have been released and said detergent into a solid phase extraction device to remove said detergent from said solution.

### Claim 43

A method of denaturing a glycoprotein or glycopeptide of interest in vitro, said method comprising incubating said glycoprotein or glycopeptide in vitro with a solution comprising effective amounts of
(a) a compound of the formula:
R—Y−.M+, in which:
R is a saturated or unsaturated straight chain aliphatic group with 8-13 carbon atoms;
Y− is a sulfate or sulfonate anion;
M+ is a cation of an element in periodic table group 1; and,
the dot indicates that the cation is ionically, not covalently, associated with the Y− anion, and either
(b) an ammonium cation detergent comprising: (i) an aliphatic chain of 8-24 carbons, (ii) a carboxylate anion covalently attached to said aliphatic chain and, (iii) a tertiary or quaternary ammonium cation ionically, not covalently, associated with the carboxylate anion, or
(b′) a molar excess of quaternary ammonium cations, or of tertiary ammonium cations, or of both.

### Claim 44 (depends on 43)

A method of claim 43, wherein said R is an aliphatic group having 12 carbons.

### Claim 45 (depends on 43)

A method of claim 43, wherein said Y− is a sulfate anion.

### Claim 46 (depends on 43)

A method of claim 43, wherein said tertiary or quaternary ammonium cation of said ammonium cation detergent is a quaternary ammonium cation.

### Claim 47 (depends on 43)

A method of claim 43, wherein said ammonium cation detergent is tetramethylammonium acetate or tetramethylammonium laurate.

### Claim 48 (depends on 43)

The method of claim 43, wherein said molar excess of said quaternary ammonium cations or tertiary ammonium cations is of quaternary ammonium cations.

### Claim 49 (depends on 46)

The method of claim 46, wherein said quaternary ammonium cation is selected from the group consisting of:

### Claim 50 (depends on 46)

The method of claim 46, wherein said quaternary ammonium cation is tetramethyl ammonium.