N-terminal modifier agents and binders for treating and analyzing peptides

Patent metadata

Field	Value
Publication number	US-12467928-B2
Application number	US-202418764943-A
Country	US
Kind code	B2
Filing date	Jul 5, 2024
Priority date	Oct 31, 2017
Publication date	Nov 11, 2025
Grant date	Nov 11, 2025

Abstract

The present disclosure relates to a metalloprotein binder that specifically binds to a N-terminally modified peptide. Also provided herein is methods and related kits for treating or analyzing a peptide using the metalloprotein binder. The methods and compositions provided herein are useful for high-throughput peptide analysis and/or sequencing.

First claim

What is claimed is:

1 . A method of analyzing a plurality of peptides, the method comprising:
(a) modifying the plurality of peptides with an N-terminal modifier agent, thereby generating a modified N-terminal amino acid (NTAA) residue on each peptide of the plurality of peptides, wherein each peptide of the plurality of peptides is attached to a solid support, each modified NTAA residue is capable of coordinating or chelating a metal cation, and wherein the N-terminal modifier agent is selected from the group consisting of:

wherein M is a metal cation binding group that comprises sulfonamide, hydroxamic acid, sulfamate, or sulfamide; the group

is a 5 or 6 membered aromatic ring containing up to three heteroatoms selected from N, O, and S as ring members, and is optionally substituted by R; R represents one or two optional substituents selected from the group consisting of F, Cl, CH 3 , CF 2 H, CF 3 , OH, OCH 3 , OCF 3 , NH 2 , N(CH 3 ) 2 , NO 2 , SCH 3 , SO 2 CH 3 , CH 2 OH, B(OH) 2 , CN, CONH 2 , CO 2 H, and CONHCH 3 ; LG is OH, ORQ, or OCC, each RQ is independently aryl or heteroaryl, each of which is optionally substituted with one or more groups selected from halo, nitro, cyano, sulfonate, carboxylate, alkylsulfonyl, and N of heteroaryl is optionally oxidized; or RQ can be —C(═O) R or —C(═O)—OR; CC is a cationic counterion; X is one of the following: O, S, Se, or NH, and wherein the modified NTAA residue on each peptide comprises one of the structures selected from the group consisting of:

wherein AA1 is a side chain of the modified NTAA residue and PP is a peptide structure except for the modified NTAA residue;
(b) contacting one or more peptides of the plurality of peptides with a set of engineered metalloprotein binders, wherein one or more engineered metalloprotein binders of the set specifically bind to a particular modified NTAA residue of the one or more peptides, wherein
each engineered metalloprotein binder of the set comprises an amino acid sequence X1-C/H/D/E-X2-C/H/D/E-X3-C/H/D/E-X4, wherein C/H/D/E is any single amino acid residue independently selected from the group consisting of amino acid residues C (Cys), H (His), D (Asp), and E (Glu); X1, X2, X3 and X4 are each any amino acid sequence independently comprising between 0 and 500 amino acid residues in length, and wherein the amino acid sequence X1-C/H/D/E-X2-C/H/D/E-X3-C/H/D/E-X4 chelates the metal cation; and
(c) generating a signal indicative of the binding of the one or more engineered metalloprotein binders to the modified NTAA residue(s) of the one or more peptides, thereby analyzing the plurality of peptides.

2 . The method of claim 1 , further comprising:
(d) removing modified NTAA residues from peptides of the plurality of peptides by an agent configured to remove modified NTAA residues, thereby exposing a new NTAA residue in each of the peptides.

3 . The method of claim 2 , further comprising: repeating steps (a)-(c) and, optionally, (d) for the new NTAA residues of the peptides.

4 . The method of claim 1 , wherein each engineered metalloprotein binder of the set is configured to specifically bind to a different modified NTAA residue of the one or more peptides.

5 . The method of claim 1 , wherein (c) further comprises determining one or more characteristics of each peptide of the one or more peptides for which the signal was generated by using predetermined binding specificities of the engineered metalloprotein binders, thereby analyzing the plurality of peptides.

6 . The method of claim 5 , further comprising determining at least partial sequence information of each peptide for which the signal was generated using the one or more determined characteristics.

7 . The method of claim 5 , wherein the set of engineered metalloprotein binders comprises at least 3 structurally different engineered metalloprotein binders each having different binding specificities towards modified NTAA residues of the one or more peptides.

8 . The method of claim 1 , wherein the engineered metalloprotein binder further comprises a detectable label.

9 . The method of claim 2 , wherein the agent configured for removing modified NTAA residues comprises an engineered enzyme.

10 . The method of claim 1 , wherein the amino acid sequence X1-C/H/D/E-X2-C/H/D/E-X3-C/H/D/E-X4 further comprises one or more amino acid sequences selected from the group consisting of: SEQ ID NO: 101 and SEQ ID NO: 102.

11 . The method of claim 10 , wherein the amino acid sequence X1-C/H/D/E-X2-C/H/D/E-X3-C/H/D/E-X4 further comprises one or more amino acid sequences selected from the group consisting of:
(A/C/D/I/S/V/Y)X(C/N)X(A/C/G/R/S/V)XX(C/F/I/L/T/V)X(C/G/K/N/V)X(F/I)(D/E/K/N/V)(D/E/F/N/Q), (A/S)XX(A/E/H/K/Q) (A/P/R/S/T)D(G/I/V)X(A/T/V) (I/L/M/N/R/V) and G(A/C/F/I/S)X(A/D/M/T)XPX(C/F/L)X(C/E/R)X(I/L/R/V), wherein X corresponds to any one of the 20 standard amino acids.

12 . The method of claim 1 , wherein each engineered metalloprotein binder comprises an amino acid sequence having at least 40% sequence identity to any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 7-SEQ ID NO: 59, SEQ ID NO: 68-SEQ ID NO: 99 and SEQ ID NO: 105-SEQ ID NO: 135.

13 . The method of claim 1 , wherein the engineered metalloprotein binder is associated with a coding tag comprising identifying information regarding the engineered metalloprotein binder.

14 . The method of claim 13 , wherein each of the plurality of peptides is associated with a recording tag comprising identifying information regarding the associated peptide or a protein from which the associated peptide is obtained from.

15 . The method of claim 14 , wherein following binding of an engineered metalloprotein binder of the set to a modified NTAA residue of a peptide of the plurality, a nucleic acid molecule is generated, using a ligation or a primer extension, which comprises the identifying information regarding the engineered binder and the identifying information regarding the peptide or a protein from which the peptide is obtained from.

16 . The method of claim 15 , wherein (c) further comprising sequencing the nucleic acid molecule, and determining one or more characteristics of the peptide by using a predetermined binding specificity of the engineered metalloprotein binder.

17 . A kit for treating a target peptide, the kit comprising:
(a) an N-terminal modifier agent selected from the group consisting of:

wherein M is a metal cation binding group that comprises sulfonamide, hydroxamic acid, sulfamate, or sulfamide; the group

is a 5 or 6 membered aromatic ring containing up to three heteroatoms selected from N, O, and S as ring members, and is optionally substituted by R; R represents one or two optional substituents selected from the group consisting of F, Cl, CH 3 , CF 2 H, CF 3 , OH, OCH 3 , OCF 3 , NH 2 , N (CH 3 ) 2 , NO 2 , SCH 3 , SO 2 CH 3 , CH 2 OH, B(OH) 2 , CN, CONH 2 , CO 2 H, and CONHCH 3 ; LG is OH, ORQ, or OCC, each RQ is independently aryl or heteroaryl, each of which is optional

Assignees

• Encodia Inc

Inventors

• Okerberg Eric
• Verespy Iii Stephen
• Klima Jason C
• Ganguly Soumya
• Miles Zachary
• Jacintho Jason Duarte
• Wise Aaron
• Bhuiya Wadud

Classifications

• G01N33/6845

  Methods of identifying protein-protein interactions in protein mixtures · CPC title

• C40B70/00

  Tags or labels specially adapted for combinatorial chemistry or libraries, e.g. fluorescent tags or bar codes · CPC title

• C40B40/10 Primary

  Libraries containing peptides or polypeptides, or derivatives thereof · CPC title

• C12Q2565/619

  being a video camera · 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