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Tandem-paired column chemistry for high-throughput proteomic exosome analysis

An analytical column and proteolysis technology, which is applied in the field of high-throughput proteomics exosome analysis with the chemical properties of series paired columns, can solve the problems of purity and sensitivity, low yield, inaccuracy, etc.

Active Publication Date: 2021-06-18
NX PRENATAL INC
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, even with samples obtained from extracellular vesicles, issues of purity and sensitivity remain
[0020] In summary, current proteomic analysis methods suffer from insufficient sensitivity and imprecision in the quantification of target peptides / proteins due to the above reasons
Additionally, current methods are time-consuming and have low yields

Method used

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  • Tandem-paired column chemistry for high-throughput proteomic exosome analysis
  • Tandem-paired column chemistry for high-throughput proteomic exosome analysis
  • Tandem-paired column chemistry for high-throughput proteomic exosome analysis

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Experimental program
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Embodiment

[0113] Frozen maternal plasma samples were used. Samples can be shipped on dry ice and stored at -80°C.

[0114] Microparticles were enriched by size exclusion chromatography (SEC) and eluted isocratically with water (RNase-free, DNase-free distilled water). The size exclusion chromatography procedure was performed using columns manufactured by AmericanBio (Natick, MA). Briefly, the columns were loaded with 10 mL of 2% Sepharose 2B (60-200um pore size) purchased from GE Healthcare Life Sciences (Marlborough, MA). The columns were stored under refrigeration. The columns were allowed to equilibrate to room temperature before they were used for exosome isolation. After equilibrating to room temperature, the column was washed with RNase-free, DNase-free distilled water. At least 0.5 mL of thawed purified plasma was applied to the column. Elute the microparticles with RNase-free, DNase-free distilled water.

[0115] Circulating particles are trapped in the column void volume ...

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Abstract

Compositions and methods for sample preparation and mass spectrometric analysis of peptide samples obtained from biological samples are provided. The compositions and methods include a tandem column system in which a trap column is in fluid contact with an analytical column such as, for example, a HPLC column. As analytes are eluted from the analytical column, they can be passed to a detector (e.g., a mass spectrometer) for peptide analysis.

Description

[0001] Statement Regarding Federally Funded Research [0002] none. [0003] References to related applications [0004] This application claims the benefit of priority to U.S. Application No. 62 / 757,922 (Rosenblatt et al.), filed November 9, 2018, the entire contents of which are incorporated herein in their entirety. [0005] Names of parties to the joint research agreement [0006] none. [0007] sequence listing [0008] none. technical field [0009] The present application is in the field of sample preparation for peptide analysis, eg analysis by high performance liquid chromatography and / or mass spectrometry. Background technique [0010] Many diagnostic procedures rely on proteomic analysis. The proteome, the unique collection of proteins present in a particular cell at a particular developmental state, can provide a wealth of information about a cell's condition and has found use in diagnostic and therapeutic strategies, especially for cancer and pregnancy dis...

Claims

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Application Information

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IPC IPC(8): B01D3/14B01D15/08B01L3/00C07K1/16G01N30/02G01N33/48H01J49/26
CPCB01D15/325B01D15/34G01N33/6851G01N33/689B01L3/502715B01L2400/0622B01L3/502753B01L2400/0487G01N30/08G01N2030/085G01N2030/8831G01N33/5076G01N33/6848G01N1/4044G01N1/405G01N30/88G01N2570/00
Inventor K·P·罗森布拉特N·哈贾M·哈立德
Owner NX PRENATAL INC
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