# US 2019/0375784 A1 · Method for pretreating protein in ex vivo body fluid

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- Number: US 2019/0375784 A1
- Document ID: US20190375784A1
- Status: Published application
- Kind: A1
- Application number: 16/462,464
- Filed: 2017-07-12
- Published: 2019-12-12
- Earliest claimed: 2016-11-21
- Assignees: Dalian Institute of Chemical Physics, Chinese Academy of Sciences
- Applicants: Dalian Institute of Chemical Physics, Chinese Academy of Sciences
- Inventors: Lihua ZHANG; Huiming YUAN; Zhigang SUI; Kaiguang YANG; Yukui ZHANG
- Web page: https://patentgrep.com/patent/US20190375784A1

## CPC

- C07K 1/145: {by extraction or solubilisation}
- C12Q 1/37: involving peptidase or proteinase
- G01N 1/4044: {by chemical techniques; Digestion; Chemical decomposition}
- G01N 1/4055: {by solubility techniques}
- G01N 30/14: by elimination of some components
- G01N 30/7233: {interfaced to liquid or supercritical fluid chromatograph (interfaces in general for introducing or extracting samples to be analysed with specially adapted mass spectrometer, see H01J49/04)}
- G01N 2001/4061: {Solvent extraction}
- G01N 2030/067: {by reaction, e.g. derivatising the sample}
- 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)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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)

## Abstract

The invention relates to a method for the treatment of body fluid proteins, by which proteins from body fluids such as blood or urine are extracted by adding a certain proportion of high molecular polymer solution under low temperature condition followed by denaturation and reduction by adding a certain concentration of surfactant and tris(2-carboxyethyl) phosphine (TCEP) under a high temperature condition. Subsequently, the iodoacetic acid brushes grafted on silica microspheres called as solid-phase alkylation reagents are added into protein solution, which can react rapidly with the protein sulfhydryl group. After centrifugation, the microspheres are obtained and repeatedly washed with methanol and buffer to remove interferences such as sugars, salts, surfactants, lipids to obtain high-purity proteins, and finally protease is added to digest proteins into peptides. After centrifugation, the peptide products are obtained, and directly analyzed by liquid chromatography-mass spectrometry (LC-MS) system. Compared with the traditional protein pretreatment method, the method has many advantages such as good anti-interference capability, easy operation and short pretreatment time.

## Claims

### Claim 1

A method for the treatment of body fluid proteins is characterized by: 1) a certain proportion of high molecular polymer solution is added to the blood or urine to extract proteins under low temperature conditions; 2) the proteins are extracted by adding a certain concentration of surfactant and reductant under a high temperature condition; 3) Solid-phase alkylation reagents are added and the proteins were obtained, finally protease is added to digest proteins into peptides.

### Claim 2 (depends on 1)

According to claim 1, the sample preparation method is characterized by: a certain proportion of high molecular polymer solution is added to the body fluids to extract proteins under low temperature conditions, the composition of the high molecular polymer might be polyethylene glycol, polyetherimide and polyvinyl alcohol with a Mr range of 500-10000, or a mixture of more than two polymer, wherein the mass ratio of high molecular polymer accounts for 1%-50% (w/w), the volume ratio of high molecular polymer solution is 1:1-10; The range of temperature is −20-10° C.

### Claim 3 (depends on 1)

According to claim 1, the sample preparation method is characterized by: a certain concentration of surfactant and protein reducing agent are added to rapidly denature the exosome protein under high temperature conditions; the surfactant can be an anionic surfactant (sodium dodecyl alcohol polyoxyethylene ether sulfate, dodecyl benzenesulfonic acid, sodium lauryl sulfate, sodium fatty acid isethionate, ammonium lauryl sulfate or a mixture of more than two anionic surfactants), a cationic surfactant(Octadecyltrimethylammonium chloride, cetyltrimethyl chloride ammonium, distearyl hydroxyethyl methyl sulfate or a mixture of more than two cationic surfactants), a zwitterionic surfactant (Dodecyl betaine, cocoyl imidazoline fatty alcohol, polyoxyethylene ether, sulfosuccinate disodium salt or a mixture of more than two zwitterionic surfactants) and a nonionic surfactant (Coconut fatty acid diethanolamide, nonylphenol polyoxyethylene ether hydroxyl synthetic alcohol, polyoxyethylene ether, C12-14 alkyl glycoside or a mixture of more than two nonionic surfactants), the concentration we used ranged from 4% to 10% (m/v), the ratio of the surfactant to the obtained solution from step 1 is 1:1-10; protein reducing agent is dithiothreitol or tris(2-carboxyethyl)phosphine or a mixture of them, the final concentration of reducing agent is 10 mM-100 mM; the temperature of protein extraction is 80-95° C.

### Claim 4 (depends on 1)

According to claim 1, the sample preparation method is characterized by: solid phase alkylating agents capable of rapidly reacting with protein thiol are added to separate exosome proteins from other small molecules to obtain high purity body fluid proteins, the solid phase alkylating agent might be iodoacetic acid-N-succinamide ester modified polymer microspheres or silica microspheres, the mass ratio of protein to solid phase alkylating agent is 1/1-1/5; The matrix of polymer microspheres might be polyacrylate, polystyrene or a mixture of them.

### Claim 5 (depends on 1)

According to claim 1, the sample preparation method is characterized by: Protease is added and incubates with the microspheres that immobilizes proteins at a certain temperature, the proteinase used may be trypsin, lysine protease, protease V8 or a mixture of more than two proteinases; the mass ratio of protein to enzyme is 1/1-1/5, and the enzymatic hydrolysis temperature is 25° C.-37° C., the enzymatic hydrolysis time is 0.5-4 hours.