-Na.n-cy-_:_-st e-i_h-e_ 040 WatersAppJicetion Notebook WatersAccQoTag TMMethodforHydrolysate AminoAcidAnalysis 120 - .s: 13.. Conditions: Column:WatersAccQ*Tag Column, 3.9 mmX150 mm D_'ivatlzati_ Chemistry 0 .Amine 1°or2 ° or Amino Acid C O _ i._ 1 + u_ _,+ Hr,.)C _ 0 ,_¢_o ColumnTemperature:37°C "_ Mobile Phase:A ternarygradient elution profileusing EluentA: AccQ*Tag EluentA EluentB: Acelonilrile EluentC: MilliQ ® water > NHS _ _:_° v 0¢--_ R2] ACK2 I_1 .._ _ / R2 + DerivatizedAmine ¢_ i(_J/_ ,--bt,_._ _1 _ _ + H20 HOO_ -,- CO, NHS _ o .._ _ _ ' = "-_ < FlowRate: 1.0 ml/mJn Detection:470 Scanning FluorescenceDetector(5_ flow celll _ "_ o a. EX:250nm EM: 395nm -1- Gain: 100 ____, 0 ,_ _ _ Filler:0.5 Injectionvolume:51.1J _ I 1 I I I I I 5 10 15 Minutes 20 25 30 35 Bothprimaryand secondaryaminesrapidly react with AQC to produce highly stable,fluorescenlderivalives. Optimized chromatographicconditionsprovide baselineor near-baselineseparationof lhe hydrolysaleaminoacids. Millipore Corporation Walers Chfomalography Division 34 Maple.Street Sample:AQC-Derivatized AminoAcid Standard (50pmoles) Milford Massachusetts 01757 508 478 2000 Objective: Details: System: To demonstratea new amino acid analysis method basedon _herapid reaction of primaryand second- Derivatizationof amino acids is an importantstep fordetection and cluantilationof the aminoacids in protein/peptide h',jdrolysates.Aminoacid labelling Withpheny[isothiocyanale(PITC)is a well-under; stoodreaclionchemistrysince il is the basisof lhe Edmandearadalion procedurefor proleinsequencincl. TheWaters PicoeTag®methoEIbasedon PITC offers rapid, highl:_erfomanceanalysisof all amino acids bul the multJpl' 'e steaderivalizalion protocol can be timeconsumincl,berivalization wilh orthophthalaldehvdeIOPA),used in Waters AutooTagTM OPA melhod, reactswilh only primary amino acids. AnotherprecolumnmethodIbasedon derivatization with dalbsylchloride hasdJfficul,ty maintaJnin_ oplimal chromatocjraphydue to tKe large inlen_ring peak produceclb_,excessreagent. Two other reagents,dans¥1chloride and fluorenyl methylchloroformate(FMOC-CI),are fluorescent melhbdswhich formmultiplederivativeswith selectedamino acids. WatersAccOeTag Systemconsisting O_a 625LC syslemequipped wilh a columnheater,717plus an/amines with AQC (AccQFluorreagenl),followed by HPLCanalysis, Waters AccQeTaclTM Systemfulfillstheresearcher's need for improved"protein/peDtidecompositional analysisby combinmaa novel precolumn derivatizationreagent spe?ificallydesianed for amino acid analysis_,_and optimal HPIL'C system configuration, includingap.ph'cationcertifiedcolumn and eluents,to provide enhanced accuracyand quantitativereproducibilily. Waters AccQeFluor_reaaent (6-aminoquinolvI-N. hydroxysuccinimi.d'yl carbamate,AQCI reactswith both primary,and secondaryamino acids within secondsan_l excessreaaent is hydrolvzedto form N-h,ydroxysuccinimide,6"-aminocluinoh*ne (AMQ) and carbbn dioxide. Thederivafized samalescan be injectedwithout furtherworkua due to }heunique fluorescentproaertiesof AMQ, theonly fluorescent byproduct of the reaction. Thisdramatically r_:k:[uces the maanitudeof the AMQ peak so that there is no interferencewith amino acid uantitation.The reaclionyields high,lystable, uorescentauinoline]abellbd ureaad_lucts.The AccQeTag 12hemistryPackage, includin_all the reagentsn;s_=_:led for _erivatization,is tes'fedand formulatedto providethelowesti_,ssibleamino acid contamination.A reversedphaseseparation with near baselineresolutionis achievedin a total runtimeof 45 minutes.TheAccQeTaa System providesresearchersa powerful,noveTmethodthat may prove usefulfor manyapplicationsin bJotechnology. Autosamplerwith heater/chiller, and 470 Scanning FluorescenceDetector.Syslemcontroland results managementwere provided by a MillenniumTM 2010 ChromatographyManager usingthe AccQeTag Application Templale. References: 1. Cohen,S. and Michaud, D., Anal. Biochem., (1993)in Press. 2. Cohen, S. A., De AntonJs,K, and Michaud, D. P., in Pressin Techniquesin ProteinChemistryIV (R.H.Angeletti, ed.)Academic Press,(1993) San Diego. R].00