Bachem Analysis of Peptides

WhatisanADS(orCofA)?

ADSstandsforAnalyticalDatasheet,CofACertificateofAnalysis.DatalistedontheADSweredeterminedfortheactualbatch.

WherearetheADSavailable?

YoucandownloadtheADSofthecurrentlyavailablelotfromtheproductentry,ifthepacksizeyouwanttoorderisavailablefromstock.

Ifnotavailableforyourorder,theADScanbedownloadedfromtheBachemwebsite,www.bachem.com.

Pleaseclickon“AnalyticalDataSheet“inthelowerpartofourwelcomepage.Youhavetoentertheproductnumberandthelotnumberwhichisimprintedonthelabelofthevialandmentionedontheinvoice.

Clickon“continue”togetyourADSaspdffile.

Whichanalyticaldatadoyouprovideforpeptides?

TherangeofanalyticaldatalistedontheADSdependsonthetypeofproduct,asmethodsforanalyzingaminoacidderivatives(andveryshortpeptides),peptides,and“non-peptide”compoundsdiffersomewhat.

AnADSissuedforapeptidecanlist:

LotnumberTypeofproduct:catalogorcustom-synthesizedProductnumberProductdescription:nameandsequenceMolecularformulaRelativemolecularmassTestsperformed:AppearanceAppearanceofsolution,solventMolecularmass,obtainedbymassspectrometryAminoacidanalysisPurity(determinedbyHPLC),upto3valuesobtainedindifferentbuffersystems“Assay”(N-content,asdeterminedbyelementalanalysis)
WhatdoesHPLCpuritymean?

Thepuritydeterminedby(analytical)HPLC(%)usingUVdetectioncorrespondstothepercentageofrequestedpeptideinrelationtothetotalamountofmaterialabsorbingat220nmi.e.thedesiredproduct,peptidicby-productsandotherimpurities.

Whatdoyoumeanby“assay(elementalanalysis)”?

“assay”istheN-contentasdeterminedbyelementalanalysis.Asthecounter-ions(acetate,trifluoroacetate..)andtheadheredwaterdonotcontainnitrogen,theN-contentcorrespondstothenetpeptidecontent.

Whatdoesgrossweightmean?

Thegrossweightofapeptidesamplecomprisestheweightofthepeptide,counter-ions,andadheredwater.

Whatdoesnetpeptidecontentmean?

Thenetpeptidecontent(NPC)isthefractionofpeptidicmaterial(i.e.therequestedpeptideandthepeptidicimpurities)relativetocounter-ionsandresidualwater.Thelatterdonotcontainnitrogen,allowingthedeterminationofthenetpeptidecontentbyelementalanalysis.Additionally,theNPCcanbedeterminedbyaminoacidanalysis.

Howtocalculatethenetweight?

Netweight=GrossweightxNetpeptidecontent(NPC)/100

Anexample:NPC85%,1mg(gross)=0.85mg(net)

Arelativelylowpeptidecontentcanbeexpectedwhenapeptidecontainsalargeproportionofbasicaminoacids,especiallyarginines.

Howtocalculatethemolarityofapeptidesolution?

Thepeptidicimpuritiesandthenetpeptidecontent(NPC)havetobetakenintoaccount,e.g.forcalculatingtheamountofsolventrequiredforpreparingasolutionofagivenconcentration.TheproportionofpeptidiccontaminantscanbedeterminedbyanalyticalHPLC.TheHPLC-puritycorrespondstothefractionofthedesiredsequence,thelowestvaluementionedontheADSshouldbeusedforcalculatingthemolarcontent:

Molaramountofdesiredpeptide=
GrossweightxNPC(%)xHPLC-purity(%)/Mol.weightx10’000

Anexample:Bradykininacetatesalt,grossweight5mg
HPLC-purity98.7%,NPC(“Assay”)85.0%,Mol.weight1060.22g/mol→net4.195mg(3.956µmol)

Thisvalueallowscalculatingconcentrations,whendissolvingthepeptide.

Whataretheremainingpeptidicimpurities?

NotallimpuritiescanberemovedbyasinglepreparativeRP-HPLCrun.Usuallyafewpeptidiccontaminantsremain,mostlydeletionsequences(peptideslackingoneormoreaminoacidsofthetargetsequence).Suchby-productsdifferonlyslightlyinpolarity,hencetheirremovalmayfail.
Truncationsequences(whichmaybegenerateddeliberatelybycappingstepstoavoidtheformationofdeletionpeptides),incompletelydeprotectedpeptidesandby-productsgeneratedduringthesynthesisorduringthefinalcleavagemayalsobefoundamongtheimpurities.

Howdoyouobtainyourmassspectra?

ByESI-MS.Additionally,MALDI-TOFandQ-TOFareroutinelyused.

Howdoyouperformanaminoacidanalysis(AAA)?

Aminoacidanalysis(AAA)isperformedbyliquid-phasehydrolysisofthepeptidewithconstant-boilinghydrochloricacid(6NHCl)followedbypre-columnderivatizationofthefreeaminoacids.ThederivatizedaminoacidsareseparatedbyRP-HPLC.Integrationoftheindividualpeaksallowsthedeterminationoftheaminoacidcompositionofthepeptidehydrolysate.However,theindolemoietyofTrpisdestroyedduringhydrolysis;theside-chainamidegroupsofAsnandGlnandthelactamcycleofPyr,willbehydrolyzedyieldingAspandGlu,respectively.Hence,theseaminoacidsarelistedasAsxandGlxintheAAAresult.

Howtoprepareapeptidesolutionofdefinedmolarconcentration?

Thepeptidicimpuritiesandthenetpeptidecontent(NPC)havetobetakenintoaccount,e.g.forcalculatingtheamountofsolventrequiredforpreparingasolutionofagivenconcentration.TheproportionofpeptidiccontaminantscanbedeterminedbyanalyticalHPLC.TheHPLC-puritycorrespondstothefractionofthedesiredsequence,thelowestvaluementionedontheADSshouldbeusedforcalculatingthemolarcontent:

Molaramountofdesiredpeptide=
GrossweightxNPC(%)xHPLC-purity(%)/Mol.weightx10’000

Anexample:Bradykininacetatesalt,grossweight5mg
HPLC-purity98.7%,NPC(“Assay”)85.0%,Mol.weight1060.22g/mol→net4.195mg(3.956µmol)

Thisvalueallowscalculatingtheamountofsolventrequiredforobtainingagivenconcentration.