US 12,510,547 B2 · Sample preparation by temperature gradient denaturation and scale-up for deep n-glycomic analysis of serum for capillary electrophoresis and CE-ESI-MS

Abstract

A sample preparation workflow to facilitate deep N-glycomics analysis of human serum by capillary electrophoresis with laser induced fluorescence (CE-LIF) detection accommodates the higher sample concentration requirement of electrospray ionization mass spectrometry connected to capillary electrophoresis (CE-ESI-MS). A temperature gradient denaturing protocol is applied on amine functionalized magnetic bead partitioned glycoproteins to avoid precipitation. This also results in the free sugar content of the serum being significantly decreased which allows PNGase F mediated release of the N-linked carbohydrates. The liberated oligosaccharides were tagged with aminopyrene-trisulfonate, utilizing a modified evaporative labeling protocol. This workflow provides appropriate amounts of material for example for use in CE-ESI-MS analysis in negative ionization mode.

Claims18

Dependent claims

A method of analyzing a serum sample, comprising:

mixing said serum sample with amine-functionalized magnetic beads so as to capture glycoproteins contained in said serum sample onto said magnetic beads, the glycoproteins comprising a glycan portion that is connected to a peptide portion;
denaturing the glycoproteins by mixing a denaturation solution with said magnetic beads and applying an increasing temperature gradient over a period of time;
releasing the glycan portion from the peptide portion in the glycoprotein captured on the magnetic bead using an enzyme to form a released glycan portion;
and
labelling the released glycan portion to form a labelled glycan portion;
and
analyzing the labelled glycan portion.

4 more elements

Dependencies are inferred from the claim text.

Facts

Application

17/641,224Filed 10 Sep 2020 · us provisional application

Priority

10 Sep 2019

Published

US 2022/0341940 A127 Oct 2022

Granted

30 Dec 2025

Term ends

Not estimatedThe source records don’t support an estimate.

Classification10 codes

G01N 33/6848
General methods of protein analysis not limited to specific proteins or families of proteins: methods of protein analysis involving mass spectrometry
G01N 33/6851
Methods of protein analysis involving mass spectrometry: methods of protein analysis involving laser desorption ionisation mass spectrometry
C12Q 1/34
Measuring or testing processes involving enzymes, nucleic acids or microorganisms ; Compositions therefor; Processes of preparing such…
G01N 1/44
Preparing specimens for investigation including physical details of chemical methods covered elsewhere, e.g. G01N33/50, C12Q: sample treatment…

Sample preparation by temperature gradient denaturation and scale-up for deep n-glycomic analysis of serum for capillary electrophoresis and CE-ESI-MS

Life of this patent

Sample preparation by temperature gradient denaturation and scale-up for deep n-glycomic analysis of serum for capillary electrophoresis and CE-ESI-MS

Life of this patent

Abstract

Claims18