Use of mild electrophiles to reduce artifacts in analyzing glycans released from glycoproteins or glycopeptides
AssigneeProZyme, Inc.
InventorMichael J. KIMZEY
Life of this patent
Estimating the term…
Abstract
The presence of mild electrophiles, such as aldehydes, during the denaturation of glycoproteins or glycopeptides and subsequent enzymatic deglycosylation reduces artifacts in subsequent analyses of the glycans released from the glycoproteins or glycopeptides.
Claims49
An in vitro method of labeling glycosylamines released from a glycoprotein or glycopeptide of interest, said method comprising:
- (a) forming a mixture of said glycoprotein or glycopeptide and a mild electrophile in a solution,
- (b) incubating said mixture in said solution at a first temperature and for a time sufficient to denature said glycoprotein or glycopeptide,
- (c) reducing said temperature of said mixture in said solution to a second temperature, which second temperature is suitable for enzymatic deglycosylation of said glycoprotein or glycopeptide,
- (d) contacting said glycoprotein or glycopeptide at said second temperature with at least one deglycosylation enzyme which releases glycans from a glycoprotein or glycopeptide as glycosylamines, thereby releasing glycans from said glycoprotein or glycopeptide as glycosylamines, and
- (e) contacting said released glycosylamines with an amine-reactive dye under conditions allowing said amine-reactive dye to label said released glycosylamines,
- thereby labeling said released glycosylamines.
A composition, comprising:
- (i) a detergent, (ii) a mild electrophile, (iii) at least one deglycosylation enzyme which releases glycans from said glycoprotein as glycosylamines, (iv) a partially or fully deglycosylated protein or peptide from which glycans have been removed by said deglycosylation enzyme, and (v) glycosylamines released from said partially or fully deglycosylated protein by said enzyme.
An in vitro method of reducing artifacts in analyzing glycans released from a glycoprotein or glycopeptide and labeled by reductive amination, said method comprising the following steps in the following order:
- (a) forming a solution comprising said glycoprotein or glycopeptide, and a mild electrophile, under conditions allowing said mild electrophile to block any nucleophilic components present in said solution or on said glycoprotein or glycopeptide,
- (b) heating said solution to a first temperature, and for a time, sufficient to denature said glycoprotein or glycopeptide,
- (c) reducing said temperature of said solution to a second, cooler temperature,
- (d) releasing glycans from said glycoprotein or glycopeptide,
- (e) labeling said glycans released from said glycoprotein or glycopeptide by reductive amination, and
- (f) analyzing said glycans labeled by said reductive amination,
- whereby said blocking of said nucleophilic components present in said solution or on said glycoprotein or glycopeptide prior to said denaturation and reductive amination reduces artifacts in said analysis of said released glycans.
A method of reducing artifacts in analyzing glycans released from a glycoprotein or glycopeptide without labeling, said method comprising the following steps in the following order:
- (a) forming a solution comprising said glycoprotein or glycopeptide, and a mild electrophile, under conditions allowing said mild electrophile to block any nucleophilic components present in said solution or on said glycoprotein or glycopeptide,
- (b) heating said solution to a first temperature, and for a time, sufficient to denature said glycoprotein or glycopeptide,
- (c) reducing said temperature of said solution to a second, cooler temperature,
- (d) releasing glycans from said glycoprotein or glycopeptide, and
- (e) analyzing said glycans labeled by said reductive amination,
- whereby said blocking of said nucleophilic components present in said solution or on said glycoprotein or glycopeptide prior to said denaturation reduces artifacts in said analysis of said released glycans.
A kit for labeling glycosylamines released from a glycoprotein or glycopeptide, said kit comprising,
- (a) a mild electrophile,
- (b) an amine-reactive dye, an agent for performing reductive amination, or both an amine-reactive dye and an agent for performing reductive amination, and
- (c) instructions on denaturing said glycoprotein or glycopeptide in the presence of said mild electrophile.
Dependencies are inferred from the claim text.