Mass spectrometry analysis of mutant polypeptides in biological samples

Patent metadata

Field	Value
Publication number	US-11428696-B2
Application number	US-202016890379-A
Country	US
Kind code	B2
Filing date	Jun 2, 2020
Priority date	Feb 1, 2010
Publication date	Aug 30, 2022
Grant date	Aug 30, 2022

Abstract

The invention relates to a method for determining the presence of at least one distinct polypeptide in a biological sample comprising contacting the biological sample with a hydrolyzing agent, wherein the hydrolyzing agent is capable of hydrolyzing the distinct polypeptide in a sequence-specific manner such that at least one distinct peptide having a predetermined peptide measured accurate mass would result if the at least one distinct polypeptide were present in the biological sample, to obtain a hydrolyzed sample; bringing the hydrolyzed sample in contact with a substrate comprising at least one immobilized binding partner, wherein the at least one immobilized binding partner is capable of specifically binding the distinct peptide; removing the hydrolyzed sample from the substrate in a manner such that the distinct peptide would remain bound to the immobilized binding partner; contacting the substrate with an elution solution, wherein the distinct peptide would dissociate from the immobilized binding partner into the elution solution; subjecting a portion of the elution solution to liquid chromatography to segregate a plurality of molecules in the portion of the elution solution to obtain sorted molecules; determining the measured accurate mass of at least one sorted molecule present in the elution solution; and determining the presence of the at least one distinct polypeptide in the biological sample when a measured accurate mass of at least one molecule is substantially equal to the predetermined peptide measured accurate mass.

First claim

What is claimed is:

1. A method for determining the presence of at least one distinct polypeptide in a biological sample comprising:
a) contacting the biological sample with a hydrolyzing agent, wherein the hydrolyzing agent is capable of hydrolyzing the distinct polypeptide in a sequence-specific manner such that at least one distinct peptide having a predetermined peptide measured accurate mass would result if the at least one distinct polypeptide were present in the biological sample, to obtain a hydrolyzed sample, wherein the at least one distinct polypeptide is a mutant polypeptide relative to a wild type polypeptide;
b) bringing the hydrolyzed sample in contact with a substrate comprising at least one immobilized binding partner, wherein the at least one immobilized binding partner is capable of specifically binding the distinct peptide;
c) removing the hydrolyzed sample from the substrate in a manner such that the distinct peptide would remain bound to the immobilized binding partner;
d) contacting the substrate with an elution solution, wherein the distinct peptide would dissociate from the immobilized binding partner into the elution solution, to obtain an elution solution comprising a plurality of molecules;
e) segregating the plurality of molecules by liquid chromatography to obtain sorted molecules;
f) determining the measured accurate mass of at least one sorted molecule present in the elution solution; and
g) determining the presence of the at least one distinct polypeptide in the biological sample when a measured accurate mass of at least one sorted molecule is substantially equal to the predetermined peptide measured accurate mass.

2. The method of claim 1 , wherein at least one standard peptide is added to the plurality of molecules, wherein the at least one standard peptide has substantially the same amino acid sequence as the at least one distinct peptide but a different measured accurate mass.

3. The method of claim 1 , wherein at least two reference peptides are added to the plurality of molecules, wherein the at least two reference peptides have known a known liquid chromatography elution order.

4. The method of claim 2 , wherein the standard peptide comprises an isotope selected from the group consisting of 15 N, 13 C, 18 O and 2 H.

5. The method of claim 2 , wherein the at least one standard peptide and the at least one distinct peptide co-segregate during liquid chromatography.

6. The method of claim 4 , wherein the presence of the at least one distinct polypeptide is confirmed by the co-segregation of the at least one standard-peptide and the at least one sorted molecule and by the difference in measured accurate mass between the at least one standard peptide and the at least one sorted molecule.

7. The method of claim 1 , wherein the liquid chromatography is selected from the group consisting of HPLC, reverse phase HPLC, and reverse phase C18 HPLC.

8. The method of claim 1 , wherein the plurality of molecules are segregated during liquid chromatography according to a feature selected from the group consisting of hydrophobicity, charge, and retention time.

9. The method of claim 2 wherein the at least one standard peptide and the at least one distinct peptide have a substantially identical retention time.

10. The method of claim 1 , wherein the difference between the mutant polypeptide and wild type polypeptide is selected from the group consisting of an addition, deletion and/or substitution of about 1 to about 10 amino acid residues and an addition, deletion and/or substitution of a post-translational modification on at least one amino acid residue.

11. The method of claim 10 , wherein the post-translational modification is selected from the group consisting of phosphorylation, acetylation, ubiquitination, and glycosylation.

12. The method of claim 1 wherein the at least one distinct peptide comprises a mutant signature.

13. The method of claim 1 , wherein the biological sample is derived from saliva, mucous, tears, blood, serum, lymph fluids, buccal cells, circulating tumor cells, mucosal cells, biopsy tissue, cerebrospinal fluid, semen, feces, plasma, urine, a suspension of cells, or a suspension of cells and viruses.

14. The method of claim 1 , wherein the biological sample comprises a number of different polypeptides selected from the group consisting of between about 5,000 to about 20,000 different polypeptides, between about 7,500 to about 15,000 different polypeptides, and up to about 100,000 different polypeptides and isoforms thereof.

15. The method of claim 1 , wherein the hydrolyzing agent is selected from the group consisting of an enzyme and a chemical.

16. The method of claim 1 , wherein the hydrolyzing agent is selected from the group consisting of cyanogen bromide, BNPS-Skatole, formic acid, trypsin, Lysine-C endopeptidase (LysC); arginine-C endopeptidase (ArgC), Asp-N, glutamic acid, endopeptidase (GluC), chymotrypsin, and combinations thereof.

17. The method of claim 1 , wherein the substrate comprises a substance selected from the group consisting of a gel matrix and polymer beads.

18. The method of claim 1 , wherein the at least one immobilized binding partner comprises a reagent selected from the group consisting of an antibody or peptide binding fragment thereof, a protein that specifically binds to the distinct peptide, an antibody or antibody fragment that binds a mutant signature peptide, and an antibody or antibody fragment that binds a wild type peptide.

19. The method of claim 1 , wherein the at least one immobilized binding partner is 1 to about 500 or 1 to at least 500 immobilized binding partners, wherein each immobilized binding partner specifically binds to a different distinct peptide.

Assignees

• Cell Signaling Technology Inc

Inventors

• Moritz Albrecht
• Rush Ii John Edward
• Polakiewicz Roberto

Classifications

• B01D15/3809

  of the antigen-antibody type, e.g. protein A, G or L chromatography · CPC title

• B01D15/325

  Reversed phase · CPC title

• G01N2570/00

  Omics, e.g. proteomics, glycomics or lipidomics; Methods of analysis focusing on the entire complement of classes of biological molecules or subsets thereof, i.e. focusing on proteomes, glycomes or lipidomes · CPC title

• B01D15/3823

  of other types, e.g. avidin, streptavidin or biotin · CPC title

• B01D15/12

  relating to the preparation of the feed · CPC title