Simon Mysling, Thomas J. D. Jørgensen University of Southern Denmark Protein Research Group June 12...
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Simon Mysling, Thomas J. D. Jørgensen University of Southern Denmark Protein Research Group June 12 th 2013 H/D exchange: New Developments in Technology
Simon Mysling, Thomas J. D. Jrgensen University of Southern
Denmark Protein Research Group June 12 th 2013 H/D exchange: New
Developments in Technology The 61 st annual ASMS conference
Electrochemical Reduction of TCEP-resistant Disulphide Bonds For
use in Hydrogen/Deuterium exchange monitored by Mass
Spectrometry
Slide 2
Disulphide bond reduction in HDX experiments Chemical reductant
Tris(2-carboxyethyl)phosphine (TCEP) Important step when analyzing
disulphide bond-containing proteins RSSR to RSH HSR Improve
proteolytic digestion Improve sequence coverage (previously
disulphide-linked peptides observable) Reduction should be rapid,
and run a quench conditions - pH 2.5, 0C Spike sample with
reductant and incubate prior to injection
Slide 3
Reduction at quench conditions using TCEP Cline, D. J.; Thorpe,
C. Biochemistry 2004, 43, 15195 pH 2.5 TCEP efficiency is severely
reduced at pH 2.5 High concentrations Accumulation in RP columns
Extensive washing
Slide 4
Three consecutive injections with 400 mM TCEP On-column
accumulation of TCEP Injection 2 Injection 3 Injection 1
Slide 5
Injection 2 Injection 3 Injection 1 On-column accumulation of
TCEP Three consecutive injections with 400 mM TCEP
Slide 6
Reduction at quench conditions using TCEP TCEP efficiency is
severely reduced at pH 2.5 High concentrations Accumulation in RP
columns Extensive washing Some disulphide bonds are less vulnerable
to TCEP reduction Difficult to analyze using HDX-MS Cline, D. J.;
Thorpe, C. Biochemistry 2004, 43, 15195 pH 2.5
Slide 7
Insulin H S H S H S H S H S H S Reduction Chain B Chain A
+
Slide 8
Reduction of Insulin using TCEP Quench conditions - 0C and pH
2.5 Insulin MH 5 + Insulin MH 4 + Insulin MH 6 + Chain B MH 4 +
Chain B MH 5 + 400 mM TCEP 10 min. incubation 400 mM TCEP 2 min.
incubation Insulin MH 5 + Insulin MH 4 + Insulin MH 6 + Insulin MH
5 + Insulin MH 4 + Insulin MH 6 +
Slide 9
Reduction of Insulin using TCEP Quench conditions - 0C and pH
2.5 Insulin MH 5 + Insulin MH 4 + Insulin MH 6 + Chain B MH 4 +
Chain B MH 5 + 400 mM TCEP 10 min. incubation 400 mM TCEP 2 min.
incubation Insulin MH 5 + Insulin MH 4 + Insulin MH 6 + Insulin MH
5 + Insulin MH 4 + Insulin MH 6 + 10 min. incubation less than 5%
reduction 50 min. Incubation ~15-20% reduction Chain B MH 4 +
Slide 10
Reduction of Insulin using TCEP Quench conditions - 0C and pH
2.5 Insulin MH 5 + Insulin MH 4 + Insulin MH 6 + Chain B MH 4 +
Chain B MH 5 + 400 mM TCEP 50 min. incubation 400 mM TCEP 10 min.
incubation 400 mM TCEP 2 min. incubation Insulin MH 5 + Insulin MH
4 + Insulin MH 6 + Insulin MH 5 + Insulin MH 4 + Insulin MH 6 + 10
min. incubation less than 5% reduction 50 min. Incubation less than
20% reduction Chain B MH 4 +
Slide 11
Reduction at quench conditions using TCEP Alternative reduction
methods could be valuable in many situations TCEP efficiency is
severely reduced at pH 2.5 High concentrations Accumulation in RP
columns Extensive washing Some disulphide bonds are less vulnerable
to TCEP reduction Difficult to analyze using HDX-MS Cline, D. J.;
Thorpe, C. Biochemistry 2004, 43, 15195 pH 2.5
Slide 12
Electrochemical reduction cell Working Electrode Solvent flow
Reference electrode 1% FA in solvent Able to reduce insulin
efficiently, using direct infusion 12 uL internal volume Running
conditions 50 bar (725 PSI) pressure limit
Slide 13
Is electrochemical reduction, at quench conditions: - Stable
and reproducible? - Going to increase back- exchange markedly?
Reduction cell Pepsin column Digestion chamber 10C Trap and
analytical column 0.2C From loop To desalting trap Injection -
Still efficient?
Slide 14
Cell off 100 L/min. Insulin MH 5 + Insulin MH 6 + Insulin MH 5
+ Insulin MH 6 + Chain B MH 5 + Chain B MH 4 + Chain B MH 5 + Chain
B MH 4 + Chain A MH 3 + Relative intensity [AU] m/z [Th] Cell on
100 L/min. Residence time: 7.2 s. Electrochemical reduction of
insulin
Slide 15
Cell off 100 L/min. Insulin MH 5 + Insulin MH 6 + Insulin MH 5
+ Insulin MH 6 + Chain B MH 5 + Chain B MH 4 + Chain B MH 5 + Chain
B MH 4 + Chain A MH 3 + Relative intensity [AU] m/z [Th] Cell on
100 L/min. Residence time: 7.2 s. Electrochemical reduction of
insulin
Slide 16
Cell off 100 L/min. Cell on 50 L/min. Residence time: 14.4 s.
Insulin MH 5 + Insulin MH 6 + Insulin MH 5 + Insulin MH 6 + Chain B
MH 5 + Chain B MH 4 + Chain B MH 5 + Chain B MH 4 + Chain A MH 3 +
Relative intensity [AU] m/z [Th] Cell on 100 L/min. Residence time:
7.2 s. Tweak reduction using the desalting flow rate
Electrochemical reduction of insulin Reduction efficiency is
dependent on residence time (Flow rate)
Slide 17
Deuterons16.114.312.9 Back-exchange28.0%36.0%42.2% Cell active
XXO Cell present XXO Buffer 0.23% FA1% FA Desalting 0.5 min. 300
ul/min 3 min. 50 ul/min 3 min. 50 ul/min Deuterons Labeled insulin
B-chain Impact on deuterium back-exchange Observed Theoretical
maximum
Slide 18
Deuterons16.114.312.9 Back-exchange28.0%36.0%42.2% Cell active
XXO Cell present XXO Buffer 0.23% FA1% FA Desalting 0.5 min. 300
ul/min 3 min. 50 ul/min 3 min. 50 ul/min Labeled insulin B-chain
Impact on deuterium back-exchange Observed Theoretical maximum
Deuterons
Slide 19
16.114.312.9 Back-exchange28.0%36.0%42.2% Cell active XXO Cell
present XXO Buffer 0.23% FA1% FA Desalting 0.5 min. 300 ul/min 3
min. 50 ul/min 3 min. 50 ul/min Impact on deuterium back-exchange
Observed Theoretical maximum Non-cooled cell in flowpath Increase
desalting time Main contributors to back-exchange Labeled insulin
B-chain Deuterons
Slide 20
- Only slightly improved deuterium back- exchange -
Considerable decrease in reduction efficiency Electrochemical
reduction was not found to alter deuteration patterns Placing the
reduction cell within a cooled environment: -Alleviated by diluting
samples 10x when quenching exchange -Other buffers could have less
dramatic effects PBS and ammonium acetate had a negative impact on
the reduction Other observations
Full deut.T6 hexamer Full exchange Undeuterated 10s exchange-in
100s exchange-in 1000s exchange-in
Slide 23
Full deut.T6 hexamer Full exchange Undeuterated 10s exchange-in
100s exchange-in 1000s exchange-in
Slide 24
Full deut.T6 hexamer Full exchange Undeuterated 10s exchange-in
100s exchange-in 1000s exchange-in
Slide 25
Full deut.T6 hexamer Full exchange Undeuterated 10s exchange-in
100s exchange-in 1000s exchange-in
Slide 26
Full deut.T6 hexamer 90 EX-1 exchange kinetics reflecting the
stability of insulin hexamers
Slide 27
Acknowledgements Finsenlaboratory, DK Michael Ploug Antec, NL
Agnieszka Kraj Biolab, DK Kim Stjerne Britta Gribsholt Sabine Amon
Protein Research Group University of Southern Denmark Novozymes, DK
Rune Salbo Funding The Lundbeck Foundation Thomas J. D. Jrgensen
Morten Beck Trelle
