Mass spectrometry-based method for simultaneous qualitative and quantitative protein citrullination analysis of complex biological samples
AssigneeWisconsin Alumni Research Foundation
InventorsLingjun Li, Yatao Shi, Zihui Li
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Abstract
The present invention discloses mass spectrometry (MS)-based methods and tagging reagents for qualitative and quantitative analysis of post-translational modified (PTM) proteins in biological and clinical samples. The present invention utilizes thiol-containing tagging reagents which are able to bind to or derivatize biomolecules, preferably post-translational modified (PTM) polypeptides such as citrullinated and homocitrullinated polypeptides. The tagging reagents are preferably biotin-derived tags able to react with a side chain of the modified polypeptides.
Claims14
A method of analyzing a target biomolecule in a sample, said method comprising the steps of:
- a) providing a sample containing the target biomolecule, wherein the target biomolecule comprises a polypeptide having a region modified by a post-translational modification (PTM);
- b) reacting the target biomolecule with a tagging reagent to generate a labeled biomolecule, wherein the reacting step comprises adding the tagging reagent with 2,3-butanedione, 2,3-butanedione monoxime, or a phenylglyoxal derivative to the target biomolecule thereby generating a derivatized polypeptide from the polypeptide having the region modified by the PTM, wherein the tagging reagent comprises a thiol group or a disulfide group able to react with the derivatized polypeptide, and wherein reacting the tagging reagent with the target biomolecule imparts a mass shift to the target biomolecule;
- c) ionizing the labeled biomolecule to form a precursor ion;
- d) detecting and analyzing the precursor ion using a mass spectrometer;
- and
- e) identifying biomolecules with mass spectrometry data;
- wherein the phenylglyoxal derivative has the formula:
- where R1 comprises a thiol group or is functional group able to react with a biotin-thiol tag.
A method of analyzing a target biomolecule in a sample, said method comprising the steps of:
- a) providing a sample containing the target biomolecule, wherein the target biomolecule comprises a polypeptide having a post-translational modification (PTM) comprising a ureido group;
- b) reacting the target biomolecule with a tagging reagent to generate a labeled biomolecule, wherein the reacting step comprises adding the tagging reagent with 2,3-butanedione, 2,3-butanedione monoxime, or a phenylglyoxal derivative to the target biomolecule thereby generating a derivatized polypeptide from the polypeptide having the region modified by the PTM comprising the ureido group, wherein the tagging reagent comprises a thiol group or a disulfide group able to react with the derivatized polypeptide, and wherein reacting the tagging reagent with the target biomolecule imparts a mass shift to the target biomolecule;
- c) ionizing the labeled biomolecule to form a precursor ion;
- d) detecting and analyzing the precursor ion using a mass spectrometer;
- and
- e) identifying biomolecules with mass spectrometry data;
- wherein the phenylglyoxal derivative has the formula:
- where R1 comprises a thiol group or is functional group able to react with a biotin-thiol tag.
Dependencies are inferred from the claim text.