Method and device for protein preparation

Patent metadata

Field	Value
Publication number	US-11009510-B2
Application number	US-201415031544-A
Country	US
Kind code	B2
Filing date	Oct 23, 2014
Priority date	Oct 24, 2013
Publication date	May 18, 2021
Grant date	May 18, 2021

Abstract

The present invention provides a method of preparing a sample comprising one or more proteins of interest, the method comprising: providing a sample comprising a population of proteins of interest solubilised with a surfactant in a medium; exposing said sample to a mild precipitant to cause precipitation of said proteins; during or after the precipitation step, bringing said sample into contact with a matrix adapted to capture said precipitated proteins and prevent excessive aggregation of precipitated protein particles; and washing the matrix with captured precipitated proteins to remove the surfactant. A sample preparation device to carry out the same is also provided.

First claim

The invention claimed is:

1. A method of preparing a sample comprising one or more proteins of interest, the method comprising the ordered steps of:
providing a sample comprising a population of proteins of interest solubilized with sodium dodecyl sulfate (SDS) in a medium, wherein the proteins are not solubilized with urea;
shearing DNA in the sample, if the sample comprises cells;
reducing disulfides and alkylating cysteines in the sample;
exposing said sample to a precipitant, wherein said precipitant solubilizes the SDS, causes precipitation of said proteins as a suspension of particles of protein, does not render the precipitated proteins insensitive to protease digestion, and wherein the precipitant comprises phosphoric acid;
adding an aqueous methanolic solution to the sample containing the precipitated proteins;
during or after the precipitation step, bringing said sample into contact with a matrix that is a depth filter having a network of pore channels adapted to capture said precipitated protein particles throughout the depth filter, thereby preventing excessive aggregation of precipitated protein particles, and further wherein said matrix does not bind the solubilized SDS;
washing the matrix containing the captured precipitated protein particles with an aqueous methanolic solution to remove the SDS; and
digesting the captured precipitated protein particles in situ using a protease.

Assignees

• Univ Leeds Innovations Ltd
• The Univ Of Leeds

Inventors

• Zougman Alexandre

Classifications

• C07K1/36

  by a combination of two or more processes of different types · CPC title

• G01N33/6842 Primary

  Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins · CPC title