Transcript Document

Electrochemical Reduction of
TCEP-resistant Disulphide Bonds
For use in Hydrogen/Deuterium exchange monitored by
Mass Spectrometry
Simon Mysling, Thomas J. D. Jørgensen
University of Southern Denmark
Protein Research Group
June 12th 2013
H/D exchange: New Developments in Technology
The 61st annual ASMS conference
Disulphide bond reduction in HDX experiments
Important step when analyzing disulphide bond-containing proteins
R–S–S–R to R–SH HS–R
Improve proteolytic digestion
Improve sequence coverage
(previously disulphide-linked peptides observable)
Reduction should be rapid, and run a quench conditions - pH 2.5, 0°C
Chemical reductant Tris(2-carboxyethyl)phosphine (TCEP)
Spike sample with reductant and incubate prior to injection
Reduction at quench conditions using TCEP
TCEP efficiency is severely reduced
at pH 2.5
High concentrations
pH 2.5
Accumulation in RP columns
Extensive washing
Cline, D. J.; Thorpe, C. Biochemistry 2004, 43, 15195
On-column accumulation of TCEP
Three consecutive injections with 400 mM TCEP
100 pmol uPAR wt (A) 100s exchange-in rep 2 - Q PBS and 400 mM TCEP for 2 min (120 ul) - Pepsin column
QS04855SM
1: TOF MS ES+
BPI
290
Injection 1
%
100
0
2.00
QS04857SM
4.00
6.00
8.00
10.00
12.00
14.00
16.00
1: TOF MS ES+
BPI
318
Injection 2
%
100
0
2.00
QS04859SM
4.00
6.00
8.00
10.00
12.00
14.00
16.00
1: TOF MS ES+
BPI
451
Injection 3
%
100
0
2.00
Time
4.00
6.00
8.00
10.00
12.00
14.00
16.00
On-column accumulation of TCEP
Three consecutive injections with 400 mM TCEP
100 pmol uPAR wt (A) 1000s exchange-in rep21 - Q PBS and 40
QS04859SM 955 (8.695) Cm (955:1084)
1: TOF MS ES+
1.97e4
647.14
100
355.03
100 pmol uPAR wt (A) 100s exchange-in rep 2 - Q PBS and 400 mM TCEP for 2 min (120
ul) - Pepsin column
QS04855SM
Injection 1
%
%
100
1: TOF MS ES+
BPI
290
0
400
0
2.00
QS04857SM
4.00
6.00
8.00
10.00
800
m/z
1200
1000
100 pmol uPAR wt (A) 1000s exchange-in rep21 - Q PBS and 40
12.00 955 (8.695)14.00
16.00 1: TOF MS ES+
QS04859SM
Cm (955:1084)
1: TOF MS ES+
647.14
1.97e4
100
BPI
318
Injection 2
%
%
100
0
2.00
QS04859SM
600
4.00
6.00
8.00
10.00
012.00
645
14.00
650
655
660
16.00
665
TOF MS
670 1: 675
680 ES+
685
m/z
690
Injection 3
BPI
100 pmol uPAR wt (A) 1000s exchange-in rep21451
- Q PBS and 40
100
QS04859SM 955 (8.695) Cm (955:1084)
%
100
1: TOF MS ES+
1.53e3
661.14
675.15
4.00
6.00
8.00
10.00
Time
663.13
%
0
2.00
12.00
14.00
16.00
677.13
679.12
0
660
665
670
675
m/z
680
Reduction at quench conditions using TCEP
TCEP efficiency is severely reduced
at pH 2.5
High concentrations
pH 2.5
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
Insulin
Reduction
Chain A
SS
HH
+
Chain B
H
S
S
H
S
H
H
S
Reduction of Insulin using TCEP
Quench conditions - 0°C and pH 2.5
Insulin
MH6+
Insulin
MH6+
Chain B
MH5+
Chain B
MH4+
Insulin
MH6+
Insulin
MH5+
400 mM TCEP
2 min. incubation
Insulin
MH4+
Insulin
MH5+
Insulin
MH4+
Insulin
MH5+
Insulin
MH4+
400 mM TCEP
10 min. incubation
Reduction of Insulin using TCEP
Quench conditions - 0°C and pH 2.5
Insulin
MH6+
Chain B
MH4+
Chain B
MH5+
Chain B
MH4+
Insulin
MH6+
Insulin
MH6+
Insulin
MH5+
400 mM TCEP
2 min. incubation
Insulin
MH4+
Insulin
MH5+
Insulin
MH4+
Insulin
MH5+
Insulin
MH4+
10 min. incubation
less than 5% reduction
50 min. Incubation
~15-20% reduction
400 mM TCEP
10 min. incubation
Reduction of Insulin using TCEP
Quench conditions - 0°C and pH 2.5
Insulin
MH6+
Chain B
MH4+
Chain B
MH5+
Chain B
MH4+
Insulin
MH6+
Insulin
MH6+
Insulin
MH5+
400 mM TCEP
2 min. incubation
Insulin
MH4+
Insulin
MH5+
Insulin
MH4+
Insulin
MH5+
400 mM TCEP
10 min. incubation
400 mM TCEP
50 min. incubation
Insulin
MH4+
10 min. incubation
less than 5% reduction
50 min. Incubation
less than 20% reduction
Reduction at quench conditions using TCEP
TCEP efficiency is severely reduced
at pH 2.5
High concentrations
pH 2.5
Accumulation in RP columns
Extensive washing
Some disulphide bonds are less vulnerable to TCEP reduction
Difficult to analyze using HDX-MS
Alternative reduction methods could be valuable in many situations
Cline, D. J.; Thorpe, C. Biochemistry 2004, 43, 15195
Electrochemical reduction cell
Reference
electrode
Able to reduce insulin
efficiently, using direct
infusion
Solvent
flow
12 uL internal volume
Running conditions
50 bar (725 PSI) pressure limit
1% FA in solvent
Working
Electrode
Injection
Trap and analytical
column – 0.2°C
Is electrochemical reduction,
at quench conditions:
Digestion
chamber – 10°C
Reduction cell
From loop
To desalting
trap
- Still efficient?
- Going to increase backexchange markedly?
- Stable and reproducible?
Pepsin column
Electrochemical reduction of insulin
Insulin
MH5+
Relative intensity [AU]
Insulin
MH6+
Chain B
MH5+
Cell off
100 μL/min.
Cell on
100 μL/min.
Chain B
MH4+
Insulin
MH6+
Insulin
MH5+
Residence time:
7.2 s.
Chain B
MH5+
Chain B
MH4+
Chain A
MH3+
m/z
[Th]
Electrochemical reduction of insulin
Insulin
MH5+
Relative intensity [AU]
Insulin
MH6+
Chain B
MH5+
Cell off
100 μL/min.
Cell on
100 μL/min.
Chain B
MH4+
Insulin
MH6+
Insulin
MH5+
Residence time:
7.2 s.
Chain B
MH5+
Chain B
MH4+
Chain A
MH3+
m/z
[Th]
Electrochemical reduction of insulin
Insulin
MH5+
Relative intensity [AU]
Insulin
MH6+
Reduction efficiency is
dependent on residence
time (Flow rate)
Chain B
MH5+
Cell on
100 μL/min.
Chain B
MH4+
Insulin
MH6+
Chain B
MH5+
Cell off
100 μL/min.
Insulin
MH5+
Residence time:
7.2 s.
Cell on
50 μL/min.
Chain B
MH4+
Chain A
MH3+
Residence time:
14.4 s.
m/z
[Th]
Tweak reduction using
the desalting flow rate
Impact on deuterium back-exchange
Labeled insulin B-chain
Deuterons 25
Theoretical
maximum
20
Observed
15
10
5
0
Deuterons
16.1
14.3
12.9
28.0%
36.0%
42.2%
Cell active
X
X
O
Cell present
X
X
O
Buffer
0.23% FA
1% FA
1% FA
Desalting
0.5 min.
300 ul/min
3 min.
50 ul/min
3 min.
50 ul/min
Back-exchange
Impact on deuterium back-exchange
Labeled insulin B-chain
Deuterons 25
Theoretical
maximum
20
Observed
15
10
5
0
Deuterons
16.1
14.3
12.9
28.0%
36.0%
42.2%
Cell active
X
X
O
Cell present
X
X
O
Buffer
0.23% FA
1% FA
1% FA
Desalting
0.5 min.
300 ul/min
3 min.
50 ul/min
3 min.
50 ul/min
Back-exchange
Impact on deuterium back-exchange
Labeled insulin B-chain
Deuterons 25
Theoretical
maximum
20
Observed
15
10
5
0
Deuterons
16.1
14.3
12.9
28.0%
36.0%
42.2%
Cell active
X
X
O
Cell present
X
X
O
Buffer
0.23% FA
1% FA
1% FA
Desalting
0.5 min.
300 ul/min
3 min.
50 ul/min
3 min.
50 ul/min
Back-exchange
Main contributors to
back-exchange
Increase desalting time
Non-cooled cell in
flowpath
Other observations
PBS and ammonium acetate had a
negative impact on the reduction
- Alleviated by diluting samples 10x
when quenching exchange
- Other buffers could have less
dramatic effects
Placing the reduction cell within a cooled
environment:
- Considerable decrease in reduction
efficiency
- Only slightly improved deuterium backexchange
Electrochemical reduction was not found to alter deuteration patterns
Insulin hexamers
T6 hexamers
Stable assemblies
R6 hexamers
Very stable assemblies
T6 hexamers
R6 hexamers
Chain B
Chain B
Undeuterated
91
686
687
688
689
690
91
686
687
688
689
690
91
686
687
688
689
91
686
687
688
91
686
687
688
Undeuterated
691
686 687 688
10s
exchange-in
689
690
691
691
686
687
688
689
690
691
690
691
686
687
688
689
690
691
689
690
691
686
687
688
689
690
691
689
690
691
686
687
688
689
690
100s exchange-in
1000s exchange-in
Full exchange
m/z [Th]
T6 hexamer
Full deut.
10s
exchange-in
100s
exchange-in
1000s
exchange-in
Full
exchange
691
T6 hexamers
R6 hexamers
Chain B
Chain B
Undeuterated
91
686
687
688
689
690
91
686
687
688
689
690
91
686
687
688
689
91
686
687
688
91
686
687
688
Undeuterated
691
686 687 688
10s
exchange-in
689
690
691
691
686
687
688
689
690
691
690
691
686
687
688
689
690
691
689
690
691
686
687
688
689
690
691
689
690
691
686
687
688
689
690
100s exchange-in
1000s exchange-in
Full exchange
m/z [Th]
T6 hexamer
Full deut.
10s
exchange-in
100s
exchange-in
1000s
exchange-in
Full
exchange
691
T6 hexamers
R6 hexamers
Chain B
Chain B
Undeuterated
91
686
687
688
689
690
91
686
687
688
689
690
91
686
687
688
689
91
686
687
688
91
686
687
688
Undeuterated
691
686 687 688
10s
exchange-in
689
690
691
691
686
687
688
689
690
691
690
691
686
687
688
689
690
691
689
690
691
686
687
688
689
690
691
689
690
691
686
687
688
689
690
100s exchange-in
1000s exchange-in
Full exchange
m/z [Th]
T6 hexamer
Full deut.
10s
exchange-in
100s
exchange-in
1000s
exchange-in
Full
exchange
691
T6 hexamers
R6 hexamers
Chain B
Chain B
Undeuterated
91
686
687
688
689
690
91
686
687
688
689
690
91
686
687
688
689
91
686
687
688
91
686
687
688
Undeuterated
691
686 687 688
10s
exchange-in
689
690
691
691
686
687
688
689
690
691
690
691
686
687
688
689
690
691
689
690
691
686
687
688
689
690
691
689
690
691
686
687
688
689
690
100s exchange-in
1000s exchange-in
Full exchange
m/z [Th]
T6 hexamer
Full deut.
10s
exchange-in
100s
exchange-in
1000s
exchange-in
Full
exchange
691
T6 hexamers
R6 hexamers
Chain B
Chain B
Undeuterated
91
686
687
688
689
690
691
686
687
688
689
690
90°
691
91
686
687
688
689
690
691
686
687
688
689
690
691
91
686
687
688
689
690
691
686
687
688
689
690
691
91
686
687
688
689
690
691
686
687
688
689
690
691
91
686
687
688
689
690
691
686
687
688
689
690
691
10s
exchange-in
100s
exchange-in
1000s
exchange-in
Full reflecting the stability
EX-1 exchange kinetics
ofexchange
insulin hexamers
m/z [Th]
T6 hexamer
Full deut.
Acknowledgements
Protein Research Group
University of Southern Denmark
Thomas J. D. Jørgensen
Morten Beck Trelle
Sabine Amon
Novozymes, DK
Rune Salbo
Antec, NL
Agnieszka Kraj
Finsenlaboratory, DK
Michael Ploug
Biolab, DK
Kim Stjerne
Britta Gribsholt
Funding
The Lundbeck
Foundation