Equipment Manual
39
©2011 Waters Corporation 1 HDX-MS at Waters
Transcript of Equipment Manual
©2011 Waters Corporation 1
HDX-MS at Waters
©2011 Waters Corporation 2
Hydrogen Deuterium Exchange MS systems
Dedicated standards and UPLC BEH separation chemistries nanoACQUITY UPLC with HDX technology Xevo G2-S QTof MS and Synapt G2-Si HDMS ProteinLynx Global Server (PLGS) and DynamX™ data
processing
©2011 Waters Corporation 3
Waters System Solution for Automated HDX
ProteinLynx Global SERVER (PLGS)
nanoACQUITY UPLC with HDX Automation technology
DynamXTM
Reliable peptic peptide ID by
MSE
Fast chromatography at 0 °C Automates HDX experiments
Accurate measurement ETD capability Ion mobility
Automated HDX data
Processing for deuteration
determination
Synapt G2-Si HDMS
©2011 Waters Corporation 4
Waters HDX Workflow at the Peptide Level
0
2
4
6
8
10
12
0.10 1.00 10.00 100.00 1000.00Rel
ativ
e D
eute
rium
Lev
el (
Da)
Time (min)
128-140
APO HOLO
peptide
OPTIONAL AUTOMATION
nanoACQUITY UPLC using 1.7 µm BEH130 C18 column for fast separation
PLGS and IDENTITYE for peptide ID
Protein labeling occurs For various times
Quenched protein is injected into HDX manager at pH 2.5
Quench pH to 2.50, temp to 0°C
Local Analysis at Peptide Level
Online Pepsin Digestion pH 2.50
Peptide map
DynamX™ Processing Deuterium
Uptake Determination
Undeuterated / H2O Deuterated / D2O
Inside HDX Manager at 0 ° C
ESI Q - Tof MSE
Protein in H 2 O, pH 7.40 at room temp
Add 20-fold excess D2O
Or ESI HDMSE
©2011 Waters Corporation 5
Measurement of average mass change over time
Time3.00 4.00 5.00 6.00 7.00 8.00
%
0
100
3.00 4.00 5.00 6.00 7.00 8.00
%
0
100
3.00 4.00 5.00 6.00 7.00 8.00
%
0
100
3.00 4.00 5.00 6.00 7.00 8.00
%
0
100
3.00 4.00 5.00 6.00 7.00 8.00
%
0
100
3.00 4.00 5.00 6.00 7.00 8.00
%
0
100
Labeling time
240 m
60 m
10 m
1 m
10 s
0 s
m/z600 605 610
%
0
100
%
0
100
%0
100
%
0
100
%
0
100
%
0
100 Uptake of deuterium from solution for different regions vary according to time
Changes in mass (distribution) correlated to conformational changes
©2011 Waters Corporation 6
Waters M-Class ACQUITY UPLC with HDX Technology
Waters next generation UPLC platform for nano to microscale separations
True UPLC separations for protein and peptide-level HDX-MS measurements
Reproducible, rapid and robust separations at 0 degrees C up to 15K psi
©2011 Waters Corporation 7
Iacob et al; Ion Mobility adds an additional dimension to mass spectrometric analysis of solution-phase hydrogen/ deuterium exchange; Rapid Commun. Mass Spectrom. 2008; 22: 2898–2904
Complex HDX Data can be Resolved by enabling Ion Mobility
©2011 Waters Corporation 8
IMS separates co-eluting labeled peptides
2 m Labeling No IMS separation
, p , , , p ( p)
m/z709 710 711 712 713 714 715 716 717 718 719
%
0
100
m/z709 710 711 712 713 714 715 716 717 718 719
%
0
100UCA064_100901_077_bsa_2m_rt_04_JA2 291 (3.567) Cm (278:294) 1: TOF MS ES+
3.99e4
UCA064_100901_077_bsa_2m_rt_04_JA 284 (3.472) Cm (278:294) 1: TOF MS ES+ 3.42e3
UCA064 100901 077 b 2 287 (3 504) C (278 306) 1 TOF MS ES
…with IMS-enabled separation
m/z709 710 711 712 713 714 715 716 717 718 719
%
0
100
UCA064_100901_077_bsa_2m 287 (3.504) Cm (278:306) 1: TOF MS ES+ 9.08e4
ASIQKFGERALKA 2+
AVEGPKL 1+
Ion Mobility Enabled
1+ 2+
©2011 Waters Corporation 9
Ion Mobility Separation No Deuterium Exposure
©2011 Waters Corporation 10
Ion Mobility Separation 100 Minute Exposure
©2011 Waters Corporation 11
Waters HDX software: DynamXTM
Automated uptake calculation
Easy view
Convenient interpretation
©2011 Waters Corporation 12
DynamX: IMS Support
Peptide Identified
10 sec
Ref
1 min
10 min
100 min
Time Course Changes in Uptake
Interfering Peptide
separated by Mobility
©2011 Waters Corporation 13
Difference plots represent uptake (Da) differences between ref and exp. A black vertical bar represents a sum of the mass differences observed for each peptide in
all time points.
Houde, et. al. J. Pharm. Sci. 2011. June 1;100(6):2071-2086
Understanding the Difference Plot
10 sec 1 min 12 min 60 min 240 min
Time-course
Black dotted line at y-axis values +1.1 Da
Blue dotted line at y-axis values +0.5 Da
Ref (IFN)
Exp (Oxidized IFN)
What it means… These displays are available in DynamX software (except confidence limit). In this data, certain peptides with the significant differences can be easily visualized. In this case, oxidized IFN revealed significant conformational changes in several peptides.
Confidence limit
Vertical bar Indicates that this peptide showed higher uptake in Oxidized IFN.
©2011 Waters Corporation 15
HDX for Comparability: Experimental ‘Butterfly Chart’
How different are the two conditions? Butterfly Chart reveals the exchange rate and deuteration incorporation in comparison for all peptides in all time points.
Where is the peptide #43? Residues 128- 140AA, localising the contribution to the difference
APO
HOLO
©2011 Waters Corporation 16
Leap HDX Automation Manager
Independent temperature zones for automated sampling processing
HDX manager
©2011 Waters Corporation 17
Automated Waters HDX-MS system
©2011 Waters Corporation 18
Enzymate Online Digestion Column
Pepsin immobilized BEH column, Waters P/N 186007233 2.1 mm X 30 mm 15,000 psi compatibility
©2011 Waters Corporation 19
HDX Phos B Check Standard
Intact phosphorylase b protein that can be used to evaluate HDX system performance , measure back exchange, and optimize methods
P/N 186006930
©2011 Waters Corporation 20
HDX/ETD Workflow
ETD/HDX can pinpoint conformational changes to a single AA residue
ETD presents a practical alternative to fragmentation techniques for high-spatial-resolution HDX studies
©2011 Waters Corporation 21
ETD with the SYNAPT® G2-Si System
Front-end ETD using glow discharge source Compatible with LC separations Permanently mounted Easy operation & maintenance
©2011 Waters Corporation 22
ETD/HDX Data can Provide Near AA Residue Spatial Resolution of D Uptake
©2011 Waters Corporation 23
Inlet Editor
©2011 Waters Corporation 24
Example Inlet Method
©2011 Waters Corporation 25
Example Inlet Method Binary Solvent Manager
Example gradient is for a 1.0 x 50 mm HSS T3 column. If using a 1.0 x 100 mm BEH column, typical flow rates are 40 to 45 µL/min Fluidic Configuration is set for Single Pump Trapping
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Example Inlet Method Binary Solvent Manager
Trapping flow for the BSM should be set to be identical as the initial gradient conditions. Sample loading time (trapping and digestion) is set on this tab.
©2011 Waters Corporation 27
Example Inlet Method Auxiliary Solvent Manager
The ASM flow rates have been increased to 1000 µL/min. Once software has been released, the ASM will be capable of different flow rates for both analytical and trapping conditions.
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Example Inlet Method HDX Manager
The HDX Manager has independent settings for the digestion and chamber temperature.
©2011 Waters Corporation 29
Example Inlet Method HDX Manager
Data channels must be activated. Errors will occur if they are not.
©2011 Waters Corporation 30
Source Conditions
©2011 Waters Corporation 31
Instrument
©2011 Waters Corporation 32
TriWave
©2011 Waters Corporation 33
TriWave DC
©2011 Waters Corporation 34
HDMSE Training April 2011
MSe and HDMSe
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Precursors IMS Separated
Ionized Precursors
Precursors Transferred to TOF MS
UPLC/HDMSE …deconvoluting chimericy
Co-Eluting Peptides
©2011 Waters Corporation 36
Precursors IMS Separated
Ionized Precursors
Precursors & Products Time Aligned
UPLC/HDMSE …deconvoluting chimericy
Co-Eluting Peptides
©2011 Waters Corporation 37
Ion mobility
UPLC/HDMSE
©2011 Waters Corporation 38
IMS MSE MS Method Settings
9 100 2000
0.4
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IMS MSE MS Method Settings
20 30
2
4
2 2 600
175
©2011 Waters Corporation 40
IMS MSE MS Method Settings
Lockspray sampling set to every 30 seconds.
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