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Simon Mysling, Thomas J. D. JrgensenUniversity of Southern DenmarkProtein Research GroupJune 12th 2013H/D exchange: New Developments in TechnologyThe 61st annual ASMS conferenceElectrochemical Reduction of TCEP-resistant Disulphide Bonds For use in Hydrogen/Deuterium exchange monitored by Mass Spectrometry

1Disulphide bond reduction in HDX experimentsChemical reductant Tris(2-carboxyethyl)phosphine (TCEP)Important step when analyzing disulphide bond-containing proteinsRSSR to RSH HSR Improve proteolytic digestionImprove sequence coverage(previously disulphide-linked peptides observable)Reduction should be rapid, and run a quench conditions - pH 2.5, 0CSpike sample with reductant and incubate prior to injection2Reduction at quench conditions using TCEP

Cline, D. J.; Thorpe, C. Biochemistry 2004, 43, 15195pH 2.5TCEP efficiency is severely reduced at pH 2.5High concentrationsAccumulation in RP columnsExtensive washingmost practitioners of HDX compensate for lack of efficiency by using higher concentrations of TCEP3Three consecutive injections with 400 mM TCEPOn-column accumulation of TCEP

Injection 2Injection 3Injection 1

Injection 2Injection 3Injection 1On-column accumulation of TCEP

Three consecutive injections with 400 mM TCEPIons can disturb the chromatogram, and we have seen examples where peptide ions we were interested in were suppressedDr.Patrick Griffin and coworkers have suggested using a lower concentration of TCEP to combat this accumulation

Reduction at quench conditions using TCEP

TCEP efficiency is severely reduced at pH 2.5High concentrationsAccumulation in RP columnsExtensive washingSome disulphide bonds are less vulnerable to TCEP reduction Difficult to analyze using HDX-MSCline, D. J.; Thorpe, C. Biochemistry 2004, 43, 15195pH 2.56Insulin

HSHSHSHS

HSHSReductionChain BChain A+7Reduction of Insulin using TCEPQuench conditions - 0C and pH 2.5

InsulinMH5+InsulinMH4+InsulinMH6+Chain B MH4+Chain B MH5+400 mM TCEP10 min. incubation400 mM TCEP2 min. incubationInsulinMH5+InsulinMH4+InsulinMH6+InsulinMH5+InsulinMH4+InsulinMH6+Reduction of Insulin using TCEPQuench conditions - 0C and pH 2.5

InsulinMH5+InsulinMH4+InsulinMH6+Chain B MH4+Chain B MH5+400 mM TCEP10 min. incubation400 mM TCEP2 min. incubationInsulinMH5+InsulinMH4+InsulinMH6+InsulinMH5+InsulinMH4+InsulinMH6+10 min. incubation less than 5% reduction 50 min. Incubation ~15-20% reductionChain B MH4+Reduction of Insulin using TCEPQuench conditions - 0C and pH 2.5

InsulinMH5+InsulinMH4+InsulinMH6+Chain B MH4+Chain B MH5+400 mM TCEP50 min. incubation400 mM TCEP10 min. incubation400 mM TCEP2 min. incubationInsulinMH5+InsulinMH4+InsulinMH6+InsulinMH5+InsulinMH4+InsulinMH6+10 min. incubation less than 5% reduction50 min. Incubation less than 20% reductionChain B MH4+based on the peak intensitiesReduction at quench conditions using TCEPAlternative reduction methods could be valuable in many situations

TCEP efficiency is severely reduced at pH 2.5High concentrationsAccumulation in RP columnsExtensive washingSome disulphide bonds are less vulnerable to TCEP reduction Difficult to analyze using HDX-MSCline, D. J.; Thorpe, C. Biochemistry 2004, 43, 15195pH 2.511

Electrochemical reduction cellWorkingElectrodeSolventflowReferenceelectrode1% FA in solventAble to reduce insulin efficiently, using direct infusion

12 uL internal volumeRunning conditions50 bar (725 PSI) pressure limitIs electrochemical reduction, at quench conditions:- Stable and reproducible?- Going to increase back-exchange markedly?

Reduction cellPepsin columnDigestionchamber 10CTrap and analyticalcolumn 0.2CFrom loopTo desalting trapInjection- Still efficient?Still efficient Running with cooled solvents and backpressure

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Cell off 100 L/min.InsulinMH5+InsulinMH6+InsulinMH5+InsulinMH6+Chain B MH5+Chain B MH4+Chain B MH5+Chain B MH4+Chain AMH3+Relative intensity [AU]m/z [Th]Cell on100 L/min.Residence time: 7.2 s.Electrochemical reduction of insulin

Cell off 100 L/min.InsulinMH5+InsulinMH6+InsulinMH5+InsulinMH6+Chain B MH5+Chain B MH4+Chain B MH5+Chain B MH4+Chain AMH3+Relative intensity [AU]m/z [Th]Cell on100 L/min.Residence time: 7.2 s.Electrochemical reduction of insulin

Cell off 100 L/min.Cell on50 L/min.Residence time: 14.4 s.InsulinMH5+InsulinMH6+InsulinMH5+InsulinMH6+Chain B MH5+Chain B MH4+Chain B MH5+Chain B MH4+Chain AMH3+Relative intensity [AU]m/z [Th]Cell on100 L/min.Residence time: 7.2 s.Tweak reduction using the desalting flow rateElectrochemical reduction of insulinReduction efficiency is dependent on residence time (Flow rate)Deuterons16.114.312.9Back-exchange28.0%36.0%42.2%Cell activeXXOCell presentXXOBuffer0.23% FA1% FA1% FADesalting0.5 min.300 ul/min3 min.50 ul/min3 min.50 ul/minDeuteronsLabeled insulin B-chainImpact on deuterium back-exchangeObservedTheoreticalmaximumNo cell, pre reduced chemicallyDeuterons16.114.312.9Back-exchange28.0%36.0%42.2%Cell activeXXOCell presentXXOBuffer0.23% FA1% FA1% FADesalting0.5 min.300 ul/min3 min.50 ul/min3 min.50 ul/minLabeled insulin B-chainImpact on deuterium back-exchangeObservedTheoreticalmaximumDeuteronsChange buffer and flowrateDeuterons16.114.312.9Back-exchange28.0%36.0%42.2%Cell activeXXOCell presentXXOBuffer0.23% FA1% FA1% FADesalting0.5 min.300 ul/min3 min.50 ul/min3 min.50 ul/minImpact on deuterium back-exchangeObservedTheoreticalmaximumNon-cooled cell in flowpathIncrease desalting timeMain contributors to back-exchangeLabeled insulin B-chainDeuterons6 percentage points14% percentage points

- Only slightly improved deuterium back-exchange- Considerable decrease in reduction efficiencyElectrochemical reduction was not found to alter deuteration patternsPlacing the reduction cell within a cooled environment:Alleviated by diluting samples 10x when quenching exchangeOther buffers could have less dramatic effectsPBS and ammonium acetate had a negative impact on the reductionOther observations20

Insulin hexamersT6 hexamersStable assemblies

R6 hexamersVery stable assemblies

Insulin hexamersT6 hexamersStable assemblies

R6 hexamersVery stable assembliesFull deut.T6 hexamer

Full exchangeUndeuterated10s exchange-in100s exchange-in1000s exchange-inThe cell enables chain-localized analysis of insulinFull deut.T6 hexamer

Full exchangeUndeuterated10s exchange-in100s exchange-in1000s exchange-inThe cell enables chain-localized analysis of insulinFull deut.T6 hexamer

Full exchangeUndeuterated10s exchange-in100s exchange-in1000s exchange-inThe cell enables chain-localized analysis of insulinFull deut.T6 hexamer

Full exchangeUndeuterated10s exchange-in100s exchange-in1000s exchange-inThe cell enables chain-localized analysis of insulinFull deut.T6 hexamer

90EX-1 exchange kinetics reflecting the stability of insulin hexamersThe cell enables chain-localized analysis of insulinAcknowledgementsFinsenlaboratory, DKMichael PlougAntec, NL Agnieszka KrajBiolab, DKKim StjerneBritta Gribsholt

Sabine AmonProtein Research GroupUniversity of Southern DenmarkNovozymes, DKRune SalboFundingThe Lundbeck FoundationThomas J. D. JrgensenMorten Beck Trelle