Compositions and methods for analysis of protein sequences and post-translational modifications

Patent metadata

Field	Value
Publication number	US-2016349269-A1
Application number	US-201515116609-A
Country	US
Kind code	A1
Filing date	Feb 4, 2015
Priority date	Feb 4, 2014
Publication date	Dec 1, 2016
Grant date	—

Abstract

The application discloses, compositions, methods, systems, and apparatuses for rapid sequence analysis of proteins, including location of post-translational modifications and disulfide bonds, is described. Limited digestion of fully denatured antibody occurs in seconds by flowing sample in 8 M urea at constant pressure through a micro column reactor containing immobilized aspergillopepsin I, resulting in a product mixture containing 3-10 kDa peptides, which is then fractionated by capillary column chromatography and analyzed by both electron transfer dissociation (ETD) and collision activated dissociation mass spectrometry. This method provides 95% sequence coverage of a mAb and detects numerous post-translational modifications. For disulfide bond location, native mAb is subjected to longer digestion times. Release of disulfide containing peptides from accessible regions of the folded protein occurs with short digestion times. The identity of peptides connected by a disulfide bond is determined using ETD and ion-ion proton transfer chemistry.

First claim

What is claimed is:

1 . A method for characterizing a protein, said method comprising:
optionally denaturing the protein; dissolving said protein in a digestion buffer; passing the digestion buffer comprising said protein through a reaction chamber comprising at least one hydrolyzing agent, wherein said protein contacts said hydrolyzing agent and is present in the chamber for a period of time (t) sufficient to produce protein fragments and digestion of said protein occurs in the chamber, wherein the passing of the digestion buffer comprising the protein through the chamber is done at an adjustable flow rate; and performing multi-segment liquid chromatography tandem mass spectrometry (LC MS/MS) to characterize the protein, wherein said characterization is selected from sequencing, identifying post-translational modifications (PTMs), and locating disulfide bonds.

2 . The method of claim 1 , wherein the protein is denatured before dissolving in the digestion buffer.

3 . The method of claim 1 , wherein the protein is exposed to the hydrolyzing agent under acidic and highly chaotropic conditions.

4 . The method of claim 3 , wherein said chaotropic conditions are urea at about 6 to about 9 Molar (M).

5 . The method of claim 4 , wherein said urea is at about 6, 7, or 8 M.

6 . The method of claim 5 , wherein said urea is at 8 M.

7 . The method of claim 4 , wherein said urea is used at a pH of about 3.0 to about 5.0.

8 . The method of claim 4 , wherein said urea is used at a pH of about 3.5 to about 4.5.

9 . The method of claim 4 , wherein said urea is used at a pH of about 3.9 or 4.0.

10 . The method of claim 3 , wherein the digested protein fragments range from about 3 kilodaltons (kDa) in mass to about 10 kDa in mass.

11 . The method of claim 3 , wherein the digested protein fragments range from about 10 kDa in mass to about 20 kDa in mass.

12 . The method of claim 3 , wherein the digested protein fragments range from about 20 kDa in mass to about 50 kDa in mass.

13 . The method of claim 1 , wherein the site of a disulfide bond is identified.

14 . The method of claim 13 , wherein said digested protein is subjected to electron transfer dissociation (ETD)/ion-ion proton transfer (IIPT) to identify the disulfide-containing fragments and N- and C-terminal sequences and localize the disulfide(s) within/connecting different domains.

15 . The method of claim 13 , wherein the digestion times for a disulfide analysis are from about 10 seconds (s) to about 20 minutes (min).

16 . The method of claim 15 , wherein the digestion times for a disulfide analysis are selected from about 12 s to about 10 min.

17 . The method of claim 13 , wherein the digestion times for a disulfide analysis are selected from the group consisting of about 12 s, 93 s, 260 s, and 740 s.

18 . The method of claim 1 , wherein the digestion times range from about 0.5 s to about 10 min.

19 . The method of claim 2 , wherein the digestion times are from about 0.5 s to about 10 minutes.

20 . The method of claim 19 , wherein the digestion times are about 0.7 s or about 5.7 s.

21 . The method of claim 1 , wherein said PTMs are selected from the group consisting of pyroglutamic acid formation, oxidation, amidation, deamidation, phosphorylation, methylation, acetylation, and glycosylation.

22 . The method of claim 1 , wherein the hydrolyzing agent is a protease.

23 . The method of claim 22 , wherein the protease is selected from the group consisting of aspergillopepsin I, LysN protease (Lys-N), LysC endoproteinase (Lys-C), endoproteinase Asp-N (Asp-N), endoproteinase Glu-C (Glu-C) and outer membrane protein T (OmpT).

24 . The method of claim 23 , wherein the protease is aspergillopepsin I (SEQ ID NO:32) or a biologically active fragment or homolog thereof.

25 . The method of claim 1 , wherein the adjustable flow rate is selected from a range of about 50 μl/min to about 4.0 μl/min.

26 . The method of claim 1 , wherein at least two proteins are characterized.

27 . The method of claim 26 , wherein said denatured protein is reduced and alkylated before dissolving is said digestion buffer.

28 . The method of claim 27 , wherein said protein is alkylated using N-(2-aminoethyl) maleimide.

29 . The method of claim 1 , wherein said protein is selected from the group consisting of an antibody, an antibody-like molecule, an antibody light chain, an antibody heavy chain, or biologically active fragments and homologs thereof.

30 . The method of claim 29 , wherein said antibody is a monoclonal antibody (mAb).

31 . The method of claim 29 , wherein said antibody is a therapeutic antibody.

32 . The method of claim 1 , wherein characterization data is obtained from said LC MS/MS performed on said protein fragments.

33 . The method of claim 1 , wherein the method is performed in a single LC-MS apparatus.

34 . The method of claim 32 , wherein the method is performed in a single run.

35 . The method of claim 32 , wherein the characterization data comprise at least 85, 90, 95, or 99% of the protein amino acid sequence.

36 . The method of claim 32 , wherein the characterization data comprise the identity of substantially all of the post-translational modifications of said protein.

37 . The method of claim 32 , wherein the characterization data comprise the location of substantially all of the post-translational modifications of said protein.

38 . The method of claim 1 , wherein said hydrolyzing agent is immobilized.

39 . The method of claim 1 , wherein a combination of electron transfer dissociation (ETD) and collision activated dissociation mass spectrometry (CAD) tandem mass spectrometry are used to characterize the resulting protein fragments.

40 . The method of claim 1 , wherein the protein is exposed to the hydrolyzing agent at a pH of about 3.0 to about 9.0.

41 . The method of claim 40 , wherein the protein is exposed to the hydrolyzing agent at a pH of about 7.0 to about 9.0.

42 . A reaction chamber as set forth in Figure S 2 useful for time-controlled digestion and size-controlled digestion of a protein before characterizing said protein using LC MS/MS, the chamber comprising:
at least one immobilized hydrolyzing agent, wherein said protein is passed through said chamber in a digestion buffer at an adjustable flow rate and said protein is present in the chamber for a period of time (t) sufficient to produce protein fragments and digestion of said protein occurs in the chamber; and
wherein when the fragments of said digested protein exit said chamber, said LC MS/MS is performed and said characterization is selected from sequencing amino acids, identifying post-translational modifications, locating post-translational modifications, and locating disulfide bonds.

43 . The reaction chamber of claim 42 , wherein said chamber is a fused silica capillary consisting of an outer diameter of 360 μm, an inner diameter of 150 μm, and a length of 8 cm.

44 . The reaction chamber of claim 42 , wherein said hydrolyzing agent is aspergill

Assignees

• Hunt Donald F
• WANG Welhan
• Zhang Lichao
• Univ Virginia Patent Foundation

Inventors

• Hunt Donald F
• Wang Weihan
• Zhang Lichao

Classifications

• G01N2440/20

  formation of disulphide bridges · CPC title

• G01N33/6848 Primary

  Methods of protein analysis involving mass spectrometry · CPC title

• G01N33/6854

  Immunoglobulins · CPC title

• G01N2440/38

  addition of carbohydrates, e.g. glycosylation, glycation · CPC title

• G01N2440/00

  Post-translational modifications [PTMs] in chemical analysis of biological material · CPC title