【WO2019211223A1】蛋白质的定性分析【专利】

((51)International Patent Classification:KR,KW,KZ,LA,LC,LK,LR,LS,LU,LY,MA,MD,ME,G01N33/68(2006.01)C07K1/16(2006.01)MG,MK,MN,MW,MX,MY,MZ,NA,NG,NI,NO,NZ,OM,PA,PE,PG,PH,PL,PT,QA,RO,RS,RU,RW,SA, (21)International Application Number:SC,SD,SE,SG,SK,SL,SM,ST,SV,SY,TH,TJ,TM,TN,PCT/EP2019/060883TR,TT,TZ,UA,UG,US,UZ,VC,VN,ZA,ZM,ZW.(22)International Filing Date:(84)Designated States(unless otherwise indicated,for every29April2019(29.04.2019)kind o f regional protection available).ARIPO(BW,GH, (25)Filing Language:English GM,KE,LR,LS,MW,MZ,NA,RW,SD,SL,ST,SZ,TZ,UG,ZM,ZW),Eurasian(AM,AZ,BY,KG,KZ,RU,TJ, (26)Publication Language:English TM),European(AL,AT,BE,BG,CH,CY,CZ,DE,DK, (30)Priority Data:EE,ES,FI,FR,GB,GR,HR,HU,IE,IS,IT,LT,LU,LV,62/664,88130April2018(30.04.2018)US MC,MK,MT,NL,NO,PL,PT,RO,RS,SE,SI,SK,SM,TR),OAPI(BF,BJ,CF,CG,Cl,CM,GA,GN,GQ,GW, (71)Applicant:BIOTAGE AB[SE/SE];Box8,75103UP¬KM,ML,MR,NE,SN,TD,TG).PSALA(SE).(72)Inventors;and Published:(71)Applicants:HOANG,Lee[US/US];3406Bella Vista—with international search report(Art.21(3))Avenue,SANTA CLARA,California95051(US).SUH,Chris[US/US];687Celadon Circle,Unit3,SAN JOSE,California95133(US).GJERDE,Douglas T.[US/US];12295Woodside Drive,SARATOGA,California95070(US).(74)Agent:BRANN AB;3690,Drottninggatan27,10359STOCKHOLM(SE).(81)Designated States(unless otherwise indicated,for everykind o f national protection available):AE,AG,AL,AM,AO,AT,AU,AZ,BA,BB,BG,BH,BN,BR,BW,BY,BZ,CA,CH,CL,CN,CO,CR,CU,CZ,DE,DJ,DK,DM,DO,DZ,EC,EE,EG,ES,FI,GB,GD,GE,GH,GM,GT,HN,HR,HU,ID,IL,IN,IR,IS,JO,JP,KE,KG,KH,KN,KP,(54)Title:QUALITATIVE ANALYSIS OF PROTEINS(57)Abstract:The present invention relates to a method for qualitativeFigure1analysis of a sample protein,which method comprises the steps of pro¬viding a water swollen gel column bed comprising bound protease and asample protein in liquid buffer,digesting the sample protein into polypep¬tides by contact with the gel bed and subjecting the polypeptides to massspectrometry(MS).The method is advantageously performed using backand forth flow of the sample protein including two or more repeats,andmay be performed at a temperature of less than about37°C,such as atemperature of less than about30°C.The invention also includes an auto¬mated method as well as a device and a kit for performing mass-basedanalysis of proteins with higher speed than the prior art while maintainingconditions that are tolerable to the protease.l a l b l cQUALITATIVE ANALYSIS OF PROTEINSField of InventionThis invention relates to protein characterization and analysis,and in particular to enzymatic cleavage of a sample protein into its composite peptides using a protease.The method of the invention efficiently provides for identification of proteins at conditions which are tolerable to the reagents used.Further,the invention also includes an automated method,a device and a kit based on and utilizing the herein-described enzymatic cleavage.BackgroundProtein characterization,identification and analysis by mass spectrometry can be accomplished by the enzymatic cleavage of the protein into its composite peptides or polypeptides.The polypeptides can be separated by ion pairing chromatography and directed into a mass spectrometer ing computer-based software,mass information on the polypeptide fragments is used to“reassemble”the original protein to identify the protein.In some cases,proteins may be analyzed directly by mass spectrometry, but in many cases,the protein is too large and must be broken into components for identification.A protease is often used for this purpose.A protease is an enzyme that performs proteolysis by cleaving peptide bonds of proteins and(poly)peptides.Trypsin,one of the most common proteases used for cleaving proteins,is a serine protease found in the digestive system of many vertebrates.Trypsin cleaves peptide chains mainly at the carboxyl side of the amino acids,lysine or arginine,except when either is followed by the amino acid,proline.Other protease enzymes that cleave proteins include Glu-C,Lys-N, Lys-C,Asp-N,Arg-C and chymotrypsin.An important part of an enzyme is called the active site.This is where specific protein molecules interact with the enzyme and the chain cleaving reaction occurs.Enzymes will。