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Methods for the rapid preparation of labeled glycosylamines from complex matrices using molecular weight cut off filtration and on-filter deglycosylation

A complex matrix, deglycosylation technology, applied in the field of rofiling, J.ProteomeRes.2014,1, can solve problems such as limited binding capacity

Pending Publication Date: 2019-04-26
WATERS TECH CORP
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these membranes are constructed from hydrophobic polymers such as PVDF, nylon, and nitrocellulose, and are limited in binding capacity because such membranes rely on adsorption-based retention

Method used

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  • Methods for the rapid preparation of labeled glycosylamines from complex matrices using molecular weight cut off filtration and on-filter deglycosylation
  • Methods for the rapid preparation of labeled glycosylamines from complex matrices using molecular weight cut off filtration and on-filter deglycosylation
  • Methods for the rapid preparation of labeled glycosylamines from complex matrices using molecular weight cut off filtration and on-filter deglycosylation

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Embodiment I

[0035] Labeled glucosylamine was obtained by rapid labeling of HeLa cell lysates (Abcam ab170197) using on-filter deglycosylation. HeLa cell lysates were mixed in a volume of 30 μL consisting of 50 mM HEPES (pH 7.9), 1% (w / v) RapiGest SF, 20 mM Dithiothreitol (DTT) to achieve a protein concentration of 0.5 mg / mL. The mixture was heated at 90° C. for 3 minutes and then diluted with 270 μL of water to form a diluted denatured complex matrix mixture. This diluted, denatured complex matrix mixture was then transferred to a 10 kDa MWCO filter unit (Pall Life Sciences AcroPrep TM Advance 96 filter plate part number 8164) and centrifuged at 4,000g for 6 minutes. The filtered concentrate was then diluted with 300 μL of water and centrifuged again at 4,000 g for 6 minutes. 20 μL of PNGase F solution (50 mIUB / mL PNGase F in 50 mM HEPES (New England BioLabs P0709), 1% (w / v) RapiGest SF pH 7.9) was dispensed onto the pooled concentrate and onto the filter. The MWCO filter unit was next...

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Abstract

Methods for preparing labeled glycosylamines from a complex matrix are provided. The methodology includes the steps of: denaturing glycoproteins in a complex matrix to form a denatured complex matrixmixture; loading the denatured complex matrix mixture onto a MWCO filtration device; adding a glycosidase enzymatic solution onto the MWCO filtration device to form a deglycosylated complex matrix mixture comprising glycosylamines; collecting glycosylamines released from the MWCO filtration device; and derivatizing glycosylamines with a rapid tagging reagent to form a plurality of labeled glycosylamines suitable for detection in various liquid chromatography systems and detectors.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to US Provisional Patent Application No. 62 / 357,552, filed July 1, 2016, which is incorporated herein by reference. Background technique [0003] The method for the analysis of glycoproteins present in complex matrices comprises the steps of deglycosylation followed by derivatization with a labeling reagent to produce derivatized glucosylamine. Under certain conditions, derivatization can be performed without isolating the glycoprotein from the biological sample. In a given case, the analysis of derivatized glucosylamine in complex matrices can continue to include high-performance liquid chromatography ("HPLC"), ultra-high performance liquid chromatography (UHPLC), mass spectrometry ("MS"), supercritical Analysis and / or detection by fluid chromatography, ultraviolet ("UV"), fluorescence ("FLR") detection, matrix-assisted laser desorption / ionization mass spectrometry ("MALDI-MS") and / or c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/76
CPCG01N30/06C12P19/28C07K1/13C07K1/36G01N33/6842B01D15/305G01N1/4077G01N1/4044G01N2001/4088G01N1/28B01D63/16G01N30/78G01N2030/027G01N2030/067
Inventor M.A.劳伯
Owner WATERS TECH CORP
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