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Preparation of samples for deep N-glycomic analysis of serum for capillary electrophoresis and CE-ESI-MS by temperature gradient denaturation and scaling

A technology of capillary electrophoresis and temperature gradient, which is applied in the direction of material analysis, material analysis, and biological material analysis by electromagnetic means, and can solve problems such as precipitation

Pending Publication Date: 2022-05-27
DH TECH DEVMENT PTE
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, with routinely used denaturing methods, precipitation occurs for serum samples larger than 10 μL, requiring more complex methods for higher sample volumes

Method used

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  • Preparation of samples for deep N-glycomic analysis of serum for capillary electrophoresis and CE-ESI-MS by temperature gradient denaturation and scaling
  • Preparation of samples for deep N-glycomic analysis of serum for capillary electrophoresis and CE-ESI-MS by temperature gradient denaturation and scaling
  • Preparation of samples for deep N-glycomic analysis of serum for capillary electrophoresis and CE-ESI-MS by temperature gradient denaturation and scaling

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Embodiment 1

[0065] The following describes a sample preparation scale-up protocol in one embodiment of deep N-glycomic analysis of human serum samples by capillary electrophoresis and CE-ESI-MS. Other modifications to this method will be within the knowledge of the skilled artisan.

[0066] Sample: 50 μL of human serum / 50 μL of mAb (30-50 mg / mL) solution.

[0067] 1. Protein Capture by Amine-functionalized Magnetic Beads

[0068] [1] Add 50 μL of sample to a 1.5 mL Eppendorf tube.

[0069] [2] Add 50 μL of 1M NaBH to the sample 3 CN (in THF).

[0070] [3] Vortex the sample for 5 seconds at maximum speed.

[0071] [4] Wait a minute.

[0072] [5] Add 1.0 mL of 0.1x PBS solution.

[0073] [6] Add 20 μL of amine functionalized magnetic beads (50 mg / mL; 1.0 μm diameter).

[0074] [7] Wait 30 minutes at room temperature (RT).

[0075] 2. Bead Washing

[0076] [8] The supernatant was removed after the magnetic beads were pulled down with a magnetic stand.

[0077] [9] Reconstitute the ...

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Abstract

A sample preparation workflow for promoting deep N-glycomics analysis of human serum by capillary electrophoresis (CE-LIF) with laser-induced fluorescence detection adapts to higher sample concentration requirements of electrospray ionization mass spectrometry (CE-ESI-MS) linked to capillary electrophoresis. A temperature gradient denaturation scheme is applied to amine functionalized magnetic bead dispensed glycoproteins to avoid precipitation. This also results in a significant reduction in free sugar content in serum, which results in the release of PNGase F mediated N-linked carbohydrates. The released oligosaccharide is labeled with aminopyrene-trisulfonate using an improved evaporation labeling scheme. The workflow provides an appropriate amount of material, for example, for CE-ESI-MS analysis performed in an anionizing mode.

Description

[0001] Relevant U.S. Application [0002] This application claims the benefit of priority from US Provisional Application No. 62 / 898,045, filed September 10, 2019, the entire contents of which are hereby incorporated by reference. technical field [0003] The teachings herein relate to methods of preparing serum samples for analysis, particularly for analysis. [0004] technical background [0005] Human serum contains glycoproteins (1) in a wide concentration range from pg / mL to mg / mL. Albumin and immunoglobulins together account for more than three quarters of the total protein content, and several abundant glycoproteins such as haptoglobin, alpha 1-antitrypsin and transferrin are present in high concentration ranges. In addition to these abundant proteins, most serum glycoproteins constitute less than 1% of serum proteins, so their analysis requires higher starting sample volumes or targeted fractionation by immunoaffinity-based preconcentration methods. Most human serum...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/543G01N27/447G01N27/68
CPCG01N33/6848G01N33/6842G01N33/54326G01N27/447G01N2333/98C12Q1/34G01N1/44G01N33/582G01N33/6851
Inventor A·格特曼M·西盖蒂
Owner DH TECH DEVMENT PTE
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