US 2026/0200971 A1 · METHODS FOR ISOLATING, DETECTING, AND QUANTIFYING INTERCELLULAR PROTEINS

Abstract

Disclosed are methods for isolating full-length intercellular proteins from a complex, multicellular environment. The disclosed methods include growing cells in the complex environment in the presence of an amino acid or alkanoic acid analog comprising a first chemical group. The first chemical group (e.g., an azide) is reactive in a bioorthogonal chemical reaction with a second chemical group (e.g., a strained alkyne) present in a cell-membrane-impermeable probe further containing a hydrophilic linker and a biotin analog moiety (e.g., desthiobiotin). The amino acid or alkanoic acid analog is incorporated into newly synthesized pericellular proteins, which are then labeled with the probe using the bioorthogonal reaction and isolated using an immobilized biotin-binding protein capture system. Captured proteins can then be retrieved in their full-length form using a mild elution process for further downstream analysis, including, e.g., protein detection and quantitation. The disclosed methods can be performed on cells grown in vitro, ex vivo, or in vivo.

Claims150

Dependent claims

A method for isolating proteins that are pericellularly localized in a multicellular system, the method comprising:

(a) culturing a plurality of cells in a cell culture system, wherein the plurality of cells are cultured in the presence of an amino acid analog functionalized with a first chemical group that is reactive in a bioorthogonal chemical reaction, and wherein the amino acid analog is incorporated into newly synthesized proteins;
(b) contacting the cell culture system with a cell-membrane-impermeable probe having the formula A-L-D,
wherein
A is a molecule comprising a second chemical group that is reactive in a bioorthogonal chemical reaction, wherein the first chemical group is reactive with second chemical group,
L is a hydrophilic linker, and
D is a molecule comprising a biotin analog moiety capable of specifically binding to streptavidin with a dissociation constant greater than about 1 pM and an off-rate kinetic constant greater than about 1×10−3 l/s, and
wherein newly synthesized proteins that are pericellularly localized are labeled with the probe by a bioorthogonal chemical reaction between the first and second chemical groups;
(c) lysing the cultured cells to generate a lysate containing the labeled proteins and other cellular and extracellular components;
(d) contacting the lysate with a capture reagent comprising a biotin-binding protein immobilized on a solid support, wherein the biotin-binding protein binds to the biotin analog moiety on the labeled proteins to yield a [labeled protein]:probe complex;
and
(e) separating the solid support from the lysate to separate the labeled proteins from the other cellular and extracellular components, thereby isolating the proteins that are pericellularly localized in the multicellular system.

8 more elements

Dependencies are inferred from the claim text.

Methods for isolating, detecting, and quantifying intercellular proteins

Life of this patent

Methods for isolating, detecting, and quantifying intercellular proteins

Life of this patent

Abstract

Claims150