# US 2019/0170761 A1 · Methods for the rapid preparation of labeled glycosylamines from complex matrices using molecular weight cut off filtration and on-filter deglycosylation > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2019/0170761 A1 - Document ID: US20190170761A1 - Status: Published application - Kind: A1 - Application number: 16/314,554 - Filed: 2017-06-19 - Published: 2019-06-06 - Earliest claimed: 2016-07-01 - Assignees: Waters Technologies Corporation - Applicants: Waters Technologies Corporation - Inventors: Matthew A. Lauber - Web page: https://patentgrep.com/patent/US20190170761A1 ## CPC - G01N 33/6842: {Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins} - B01D 15/305: {Hydrophilic interaction chromatography [HILIC]} - B01D 63/16: Rotary, reciprocated or vibrated modules - C07K 1/13: Labelling of peptides - C07K 1/36: by a combination of two or more processes of different types - G01N 1/28: Preparing specimens for investigation {including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q}(mounting specimens on microscopic slides G02B21/34; means for supporting the objects or the materials to be analysed in electron microscopes H01J37/20 {; laboratory gas handling apparatus B01L5/00}) - G01N 1/4077: {by other techniques involving separation of suspended solids} - G01N 30/06: Preparation - G01N 30/78: using more than one detector - G01N 1/4044: {by chemical techniques; Digestion; Chemical decomposition} - G01N 2001/4088: {filtration} - G01N 2030/027: {Liquid chromatography} - G01N 2030/067: {by reaction, e.g. derivatising the sample} - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) - B01D: SEPARATION (separating solids from solids by wet methods B03B, B03D; by pneumatic jigs or tables B03B; by other dry methods B07; magnetic or electrostatic separation of solid materials from solid materials or fluids, separation by high-voltage electric fields B03C; centrifuges, vortex apparatus B04; presses per se for squeezing-out liquid from liquid-containing material B30B9/02; treatment of water C02F, e.g. softening by ion-exchange C02F1/42; {arrangements of air intake cleaners in gas turbine plants F02C7/05} ; arrangements or mounting of filters in air-conditioning, air-humidification or ventilation F24F13/28) - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) ## 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 matrix mixture; 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. ## Claims ### Claim 1 A method for preparing labeled glycosylamines from a complex matrix, comprising the steps of: denaturing glycoproteins in a complex matrix to form a denatured complex matrix mixture; loading the denatured complex matrix mixture onto a MWCO filtration device; adding a glycosidase enzymatic solution onto the MWCO filtration device wherein the glycoproteins on the MWCO filtration device are deglycosylated and 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. ### Claim 2 (depends on 1) The method of claim 1, wherein the MWCO filtration device is heated. ### Claim 3 (depends on 1) The method of claim 1, wherein the MWCO filtration device is incubated. ### Claim 4 (depends on 1) The method of claim 1, wherein the denatured complex matrix mixture is loaded onto the MWCO filtration device by a liquid handling system. ### Claim 5 (depends on 1) The method of claim 1, further comprising the step of diluting the denatured complex matrix mixture. ### Claim 6 (depends on 1) The method of claim 1, further comprising the step of centrifuging the denatured complex matrix mixture. ### Claim 7 (depends on 1) The method of claim 1, further comprising the step of diluting the deglycosylated complex matrix mixture. ### Claim 8 (depends on 1) The method of claim 1 wherein the glycosylamines flow through the MWCO filtration device to filtrate collection device. ### Claim 9 (depends on 1) The method of claim 1, wherein the plurality of labeled glycosylamines are detected in a liquid chromatography system. ### Claim 10 (depends on 1) The method of claim 1, wherein the MWCO filtration device is a 96-well filter plate. ### Claim 11 A kit for preparing labeled glycosylamines comprising a MWCO filtration device, a rapid tagging reagent, a denaturing solution and an enzymatic solution, wherein the denaturing solution denatures glycoproteins in a complex matrix to form a denatured complex matrix mixture, the MWCO filtration device is configured to filter the denatured complex mixture, and the enzymatic solution is mixed and incubated with the denatured complex matrix mixture on the filtration device to yield glycosylamines, the rapid tagging reagent derivatizes the glycosylamines which are detectable in liquid chromatography, capillary electrophoresis or mass spectrometry systems. ### Claim 12 A liquid chromatography system comprising a MWCO filtration device, a fluorescence detection device and a mass spectrometric detection device, wherein the MWCO filtration device is configured to produce glycosylamines which flow through the filtration device and are collected for derivatization by a rapid tagging reagent and subsequent fluorescence detection in the fluorescence detection device and/or mass spectrometric detection in the mass spectrometric detection device. ### Claim 13 A liquid handling system comprising a MWCO filtration device, wherein the MWCO filtration device is configured to produce glycosylamines which flow through the filtration device and are collected for derivatization by a rapid tagging reagent and subsequent fluorescence detection in a fluorescence detection device and/or mass spectrometric detection in a mass spectrometric detection device.