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US 2006/0141528 A1Published application
Compositions and methods for quantification of serum glycoproteins
OwnerRudolf Aebersold, Hui Zhanginventors
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Abstract
The invention provides compositions and methods for identifying and/or quantifying glycopolypeptides from human serum or plasma. The compositions and methods include a plurality of standard peptides containing glycosylation sites determined for human serum/plasma proteins.
Claims50
A method for identifying glycopolypeptides in a serum or plasma sample, comprising:
- (a) derivatizing glycopolypeptides in said sample;
- (b) immobilizing said derivatized sample glycopolypeptides to a solid support;
- (c) cleaving said immobilized sample glycopolypeptides, thereby releasing non-glycosylated peptide fragments and retaining immobilized sample glycopeptide fragments;
- (d) labeling said immobilized sample glycopeptide fragments with an isotope tag;
- (e) releasing said sample glycopeptide fragments from said solid support, thereby generating released sample glycopeptide fragments;
- (f) adding to said released sample glycopeptide fragments a plurality of standard peptides selected from peptides containing the glycosylation sites referenced as SEQ ID NOS:
- 1-3482, wherein said standard peptides correspond to peptides derivatized as in step (a), cleaved as in step (c), and released as in step (e), and wherein said standard peptides are differentially labeled with a corresponding isotope tag as used in step (d);
- (g) analyzing said released sample glycopeptide fragments using mass spectrometry;
- and
- (h) identifying released sample glycopeptide fragments that correspond to standard peptides added in step (f).
7 more elements
A method for identifying glycopolypeptides in a serum or plasma sample, comprising:
- (a) immobilizing said sample glycopolypeptides to a solid support;
- (b) cleaving said immobilized sample glycopolypeptides, thereby releasing non-glycosylated peptide fragments and retaining immobilized sample glycopeptide fragments;
- (c) labeling said immobilized sample glycopeptide fragments with an isotope tag;
- (d) releasing said sample glycopeptide fragments from said solid support, thereby generating released sample glycopeptide fragments;
- (e) adding to said released sample glycopeptide fragments a plurality of standard peptides selected from peptides containing the glycosylation sites referenced as SEQ ID NOS:
- 1-3482, wherein said standard peptides correspond to peptides cleaved as in step (b), and released as in step (d), and wherein said standard peptides are differentially labeled with a corresponding isotope tag as used in step (c);
- (f) analyzing said released sample glycopeptide fragments using mass spectrometry;
- and
- (g) identifying released sample glycopeptide fragments that correspond to standard peptides added in step (e).
6 more elements
A method for identifying and quantifying glycopolypeptides in a control serum or plasma sample, comprising:
- (a) derivatizing glycopolypeptides in a said sample;
- (b) immobilizing said derivatized sample glycopolypeptides to a solid support;
- (c) cleaving said immobilized sample glycopolypeptides, thereby releasing non-glycosylated peptide fragments and retaining immobilized sample glycopeptide fragments;
- (d) labeling said immobilized sample glycopeptide fragments with an isotope tag;
- (e) releasing said sample glycopeptide fragments from said solid support, thereby generating released sample glycopeptide fragments;
- (f) adding to said released sample glycopeptide fragments a plurality of standard peptides selected from peptides containing the glycosylation sites referenced as SEQ ID NOS:
- 1-3482, wherein said standard peptides correspond to peptides derivatized as in step (a), cleaved as in step (c), and released as in step (e), and wherein said standard peptides are differentially labeled with a corresponding isotope tag as used in step (d);
- (g) analyzing said released sample glycopeptide fragments using mass spectrometry;
- (h) identifying released sample glycopeptide fragments that correspond to standard peptides added in step (f);
- and
- (i) quantifying the amount of said sample glycopeptide fragments identified in step (h).
8 more elements
A method for identifying one or more diagnostic markers for a disease, comprising:
- (a) derivatizing glycopolypeptides in a serum or plasma sample from an individual having a disease;
- (b) immobilizing said derivatized sample glycopolypeptides to a solid support;
- (c) cleaving said immobilized sample glycopolypeptides, thereby releasing non-glycosylated peptide fragments and retaining immobilized sample glycopeptide fragments;
- (d) labeling said immobilized sample glycopeptide fragments with an isotope tag;
- (e) releasing said sample glycopeptide fragments from said solid support, thereby generating released sample glycopeptide fragments;
- (f) adding to said released sample glycopeptide fragments a predetermined amount of a plurality of standard peptides selected from peptides containing the glycosylation sites referenced as SEQ ID NOS:
- 1-3482, wherein said standard peptides correspond to peptides derivatized as in step (a), cleaved as in step (c), and released as in step (e), and wherein said standard peptides are differentially labeled with a corresponding isotope tag as used in step (d);
- (g) analyzing said released sample glycopeptide fragments using mass spectrometry;
- (h) identifying released sample glycopeptide fragments that correspond to standard peptides added in step (f);
- (i) quantifying the amount of said sample glycopeptide fragments identified in step (h);
- and
- (j) comparing the amount of said sample glycopeptide fragments determined in step (i) to the amount of the same glycopeptide fragments determined in a normal serum or plasma sample.
9 more elements
A composition comprising a plurality of peptides containing the glycosylation sites referenced as SEQ ID NOS:
- 1-3482, wherein said peptides each correspond to peptide fragments derived by cleavage of polypeptides using the same cleavage reagent.
A kit comprising a plurality of peptides containing the glycosylation sites sh referenced as SEQ ID NOS:
- 1-3482, wherein said peptides each correspond to peptide fragments derived by cleavage of polypeptides using the same cleavage reagent.
Dependencies are inferred from the claim text.