US-12625144-B2 - Chemoselective sensitivity booster for tagging a peptide, peptide conjugate, or similar reactive molecule

US12625144B2US 12625144 B2US12625144 B2US 12625144B2US-12625144-B2

Abstract

The invention pertains to chemoselective sensitivity booster for tagging a peptide, peptide conjugate, or similar reactive molecule for analysis of a peptide, protein, antibody, protein bioconjugate, antibody bioconjugate, and similar analytes. The sensitivity booster comprises of sp2 or sp3 nitrogen centers in combination with hydrophobic carbon chains linked with an electrophile or nucleophile for attachment with a peptide, peptide conjugate, or molecules with similar reactivity.

Inventors

Vishal RAI
Rohith SINGUDAS
Neelesh Chandrakant REDDY

Assignees

Indian Institute Of Science Education And Research Bhopal

Dates

Publication Date: 20260512
Application Date: 20200604
Priority Date: 20190605

Claims (8)

1 . A chemoselective sensitivity booster for tagging a peptide or peptide conjugate for analysis of a peptide, protein, protein bioconjugate, and antibody, wherein the chemoselective sensitivity booster comprises: sp2 or sp3 nitrogen centers in combination with hydrophobic carbon chains linked with an electrophile or a nucleophile, wherein the chemoselective sensitivity booster is selected from the group consisting of 2,5-dioxopyrrolidin-1-yl picolinate, 2,5-dioxopyrrolidin-1-yl 4-(dibenzylamino)benzoate, 2,5-dioxopyrrolidin-1-yl 3-(benzyl(pyridin-2-ylmethyl)amino)propanoate, 2,5-dioxopyrrolidin-1-yl 3-(bis(pyridin-2-ylmethyl)amino)propanoate, 2,5-dioxopyrrolidin-1-yl 4-(4-((dibenzylamino)methyl)-1H-1,2,3-triazol-1-yl)benzoate, 2,5-dioxopyrrolidin-1-yl-4-((6-(1H-imidazol-1-yl)hexyl)oxy)benzoate, 1-(6-(4-(((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)phenoxy)hexyl)pyridin-1-ium bromide, 1-benzyl-3-(6-(4-(((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)phenoxy)hexyl)-1H-imidazol-3-ium bromide, 4-(dimethylamino)-1-(6-(4-(((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)phenoxy) hexyl)pyridin-1-ium bromide, N-(bis(dimethylamino)methylene)-N-butyl-6-(4-(((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)phenoxy)hexan-1-aminium bromide, and 6-(aminooxy)-N-(bis(dimethylamino)methylene)-N-(6-phenoxyhexyl)hexan-1-aminium bromide.
2 . The chemoselective sensitivity booster as claimed in claim 1 , wherein a sensitivity of tagged peptide detection is up to attomolar concentration as measured by mass spectrometry.
3 . The chemoselective sensitivity booster as claimed in claim 1 , wherein the conjugation of the booster is at either to a polar or a non-polar peptide.
4 . The chemoselective sensitivity booster as claimed in claim 1 , wherein a signal enhancement of the tagged peptides has a mass shift of Δm=374 Da with the conjugation of sensitivity booster in mass spectrometry.
5 . The chemoselective sensitivity booster as claimed in claim 1 , wherein the conjugation of the chemoselective sensitivity booster is at ε-amine of C-terminus Lys in a peptide or with an N-terminus α-amine.
6 . The chemoselective sensitivity booster as claimed in claim 1 , wherein a sequence coverage of the tagged peptides in peptide mapping is 75-100%.
7 . The chemoselective sensitivity booster as claimed in claim 1 , wherein a sensitivity and ease of detection of fragments in a tandem mass spectrometry (MS-MS) of peptide is improved.
8 . The chemoselective sensitivity booster as claimed in claim 1 , wherein the conjugation of sensitivity booster is for peptide detection in proteins, protein bioconjugates, antibodies including monoclonal antibody (mAb), and antibody conjugates.

Description

SEQUENCE LISTING The present application is being filed with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled “Sequence Listing_0001.txt” created on Feb. 2, 2026, which is 20,281 bytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety. FIELD OF INVENTION The invention pertains to the field of protein chemistry and specifically to chemoselective sensitivity booster for tagging a peptide, peptide conjugate, or similar reactive molecule for analysis of a peptide, protein, antibody, protein bioconjugate, antibody bioconjugate, and similar analyte. BACKGROUND OF INVENTION Protein bioconjugation caters to the diverse requirements of biophysical chemistry, biochemistry, protein-targeted, and protein-based therapeutics. In recent years, the chemical technologies for precise labelling of native proteins has witnessed remarkable growth. However, the unambiguous characterization of protein bioconjugates poses several challenges and has been responsible for slowing the developments. Mass spectrometry (MS) has become the first choice for analysis of protein bioconjugates due to the superior sensitivity and excellent ability to provide structural information. Here, the first step involves the estimation of their mass to charge ratio (m/z). Subsequently, the protein is digested by a proteolytic enzyme, and the m/z of peptides is recorded for their mapping. The peptide(s) with a label is selected and taken forward for the fragmentation and sequenced by tandem mass spectrometry (MS-MS). The latter allows the identification of the site of labelling in a protein bioconjugate. The sensitivity and accuracy of peptide mapping and its MS plays a defining role in the success of such protein sequencing. In this perspective, there have been efforts to improve the technologies associated with hardware, software, and chemical derivatization. In the attempts of single-site chemical modification, the protein with an additional mass of one label is assumed to be modified at a single residue. Further, the validation comes from the identification of a single peptide with the label. Unfortunately, the MS-MS does not map a considerable part of the peptides due to the partial digestion, limited ionization, or suppression from other ions. Hence, the absence of labelled peptide in a digested protein bioconjugate is common and adds to the analytical challenges. The limitations of the existing knowledge in the art is that, even if we identify a single labelled peptide, the confirmation of homogeneity comes with an assumption that the un-detected peptides are not labelled. Besides, the MS-MS does not map all the ions even for the detected labelled peptide. Hence, it becomes essential to develop tools that can enhance the detection of peptides and its subsequent fragments. OBJECT OF THE INVENTION An object of the invention is for a chemo-selective sensitivity booster for tagging a peptide, peptide conjugate, or similar reactive molecule for analysis of a peptide, protein, protein bioconjugate, antibody, and similar analyte, for enhancing the sensitivity of peptide detection up to attomolar concentration by mass spectrometry. An object of the invention is for a sensitivity booster which comprises of sp2 or sp3 nitrogen centers in combination with hydrophobic carbon chains linked with an electrophile or nucleophile for attachment with a peptide, peptide conjugate, or molecules with similar reactivity. Another object of the invention is to enhance the primary sequence coverage during the peptide mapping by mass spectrometry. Another object of the invention is for a method to simplify and enhance the MS-MS fragmentation pattern enabling the unambiguous sequencing of peptides, proteins, antibodies and protein bioconjugates. DESCRIPTION OF DRAWINGS AND FIGURES FIG. 1a represents a few of the potential sensitivity boosters of the invention conjugated with model peptide Gly-Phe (GF, 1) 2a-2k. 1b represents the relative intensity of the dipeptide and the sensitivity boosters of the invention conjugated with model peptide (2a-2k) in ESI-MS. FIG. 2a represents improvement in the detection peptides for tryptic digest of cytochrome C by tagging with the sensitivity booster. FIG. 2b represent simplification and improvement in the MS-MS fragmentation pattern for cytochrome C. FIG. 3 depicts that the sensitivity booster improves the peptide mapping of trastuzumab, a monoclonal antibody for directed cancer chemotherapeutics. FIG. 4a depicts the tagging with sensitivity booster improves the detection of heterogeneity in the bioconjugation of a protein. FIG. 4b depicts selective enhancement of the peptides with the modified sites by sensitivity booster (9). FIG. 5: (a) MS of VGVAPG-NH2(SEQ ID NO: 1), (b) MS of Tag-VGVAPG-NH2 (SEQ ID NO: 1), (c) MS-MS of VGVAPG-NH2 (SEQ ID NO: 1), (d) MS-MS of Tag-VGVAPG-NH2 (SEQ ID NO: 1). FIG. 6: (a) MS of AEDDVEDY