New Developments in DynamX 3 - Waters Corporation
Transcript of New Developments in DynamX 3 - Waters Corporation
©2014 Waters Corporation 1
Alex Muck, PhDHigh-Resolution System Support Group
New Developments in DynamX 3.0..and insights into pDRE
©2014 Waters Corporation 2
Prototype
HDX-MS Collaboration with Prof. John R. Engen
(Northeastern University)
CommercialProduct Launch
2005
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1.0
UPLC for HDX2014
2.0 3.0
ACQUITY M-Class UPLC with HDX-MS Technology
Early Access Beta Program
HDX-Director Software
Waters HDX Development Waters HDX Development TimelineTimeline
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DynamXDynamX –– Automate Automate PProcessing, rocessing, Eliminate Eliminate HHuman uman EError, and Save rror, and Save TTimeime
Data processing time
Manual
Manual Calculationof Deuterium Uptake
Semi-Automated
Help via Export to Excel Macro etc.
FullyAutomated
DynamX
Full Month 2 Weeks 1 – 2 Days
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Waters HDX System EnhancementsWaters HDX System EnhancementsNext Generation System for 2014Next Generation System for 2014
Analytical Standards (ASR)
Training & Support
HDX MS Workflow
Sample Preparation
Liquid Chromatography
Online Pepsin Digestion
Q-TOF MS Informatics
ACQUITY UPLC ®M-Class
MSE
IMS ETD
DynamX™3.0Automation
HDX Director Enzymate™
Pepsin Column
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DynamXDynamX 3.0 What’s 3.0 What’s NewNew
Automates processing of intact protein, peptide digest, and electron transfer dissociation (ETD) fragment level HDX data
Supports ETD fragment analysis for residue-specific structural information
Communicates HDX uptake and sample differences through enhanced coverage map and heat map displays
Selective displays of ion mobility spectra (HDMSE) data for more in-depth protein coverage
Exports to PyMOL (Schrödinger) for structural modeling of HDX-MS data
20 active beta testing sites
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Ease of UseRight from the Start
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MS Files BrowserMS Files Browser
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MS Files BrowserMS Files Browser
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MS Files BrowserMS Files Browser
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MS Files MS Files BrowserBrowserGlobal, Local, Fragment Global, Local, Fragment
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Enhanced VisualizationCoverage Map, Heat Map, Difference Plot
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Coverage Map Coverage Map –– v3.0v3.0Relative Fractional Uptake is DisplayedRelative Fractional Uptake is Displayed
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Coverage Map OverlaysCoverage Map Overlays
Absolute measurement (Daltons). Difference in relative uptake between
two states for each peptide. Using this color palette the magnitude of
difference is related to intensity; color shows which state has greater uptake (red = Lispro, blue = Insulin).
Relative Uptake Difference
A percentage based measurement. Difference in fractional uptake between
two states for each peptide. Using this color palette the magnitude of
difference is related to intensity; color shows which state has greater uptake (red = Lispro, blue = Insulin).
Fractional Uptake Difference
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Coverage Map OverlaysCoverage Map OverlaysMultiple ExposuresMultiple Exposures
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State 1
State 2
Butterfly plot - difference chart
Coverage map - fractional uptake difference
Same plotThe only difference is the palette: one is rainbow and the other is blue-red
Enhanced Visualization Enhanced Visualization -- Coverage Map Coverage Map Rainbow and BlueRainbow and Blue--WhiteWhite--Red PalettesRed Palettes
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Fractional Uptake Difference Fractional Uptake Difference Boxes Boxes if if Preferred Preferred for for VisualizationVisualization
Coverage map – clearly defines the point mutation
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Heat Map Heat Map –– v3.0v3.0Relative Fractional Uptake is DisplayedRelative Fractional Uptake is Displayed
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Heat Map Heat Map –– v3.0 v3.0 NEW Difference ModeNEW Difference Mode
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Extending the Dynamic Extending the Dynamic Range in Range in HDMSeHDMSe
pDRE – Programmable Dynamic Range Enhancement in continuum mode
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UPLC - MSE
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Conventional UPLC separation and orthogonal ion mobility separation
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Conventional UPLC separation and orthogonal ion mobility separation
Separation of intense ions into mobility peaks can lead to signal saturation on detector and relative decrease of BPI signal in displayed chromatogram..
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UPLC – HDMSE DRE
Illustration of BPI chromatogram intensity profile obtained using HDMSe DRE
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Pusher Detector
pp--DRE Alternating Transmission SwitchDRE Alternating Transmission Switch
DRE lens
Gas cell
SCAN 1- High transmission
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Pusher Detector
pp--DRE Alternating Transmission SwitchDRE Alternating Transmission Switch
DRE lens
Gas cell
SCAN 1- Low transmission (5%)
Changing p-DRE lens duty cycle allows precise control of transmission.
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?%
m/z
High Transmission
%
LowTransmission
pp--DRE Alternating Transmission SwitchDRE Alternating Transmission Switch
m/z
Stitched via Software
%
m/z
Composite spectrum
Dynamic range = 2x105
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Stitching IMS dataStitching IMS data
LCMS_MobDRE_113.raw:2
LCMS_MobDRE_113.r aw : 2
LCMS_MobDRE_114.raw:2
LCMS_MobDRE_114.raw : 2
Every saturated data pointreplaced by unsaturated point
High Transmission
LowTransmission
Composite 3D spectrum
Dynamic range = 2x103
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BPI Scale EnhancementBPI Scale Enhancement
no DRE pDRE
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pDREpDRE
Acquisition of IMS data in HDMSe mode using dynamic rage enhancement can be now performed routinely using SCN 916 software.
The impact on dynamic range is seen, where the true BPI intensity profile of the complex species analysed can be seen.
Also using the mobility data viewer, the true enhancement to peak capacity produced using ion mobility can be seen.
Collision cross section measurement is performed routinely in DRE mode.
Excellent CCS reproducibility has been illustrated in matrix and for solvent standards.
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HDMS
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HDMS as Visualized BeforeHDMS as Visualized Before
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HDMS as Visualized BeforeHDMS as Visualized Before
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New HDMS VisualizationNew HDMS Visualization
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ETDSite SpecificCumulative
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Site Specific Deuterium IncorporationSite Specific Deuterium IncorporationNavigation OptionsNavigation Options
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Resolved Deuterium IncorporationResolved Deuterium Incorporation
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ETDETDState Comparison Bound State Comparison Bound vsvs UnboundUnbound
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ETDETDDifference Difference -- Bound Bound vsvs UUnboundnbound
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Deuterium Content Deuterium Content of of zz--ionsionsUnbound vs. BoundUnbound vs. Bound
Bound (EGFR+Adnectin)TQLG (15-18) is known region of interaction. (Ramamurthy, et. al., Structure, 2012, 20, 259-269
Significant uptake difference was observed between bound and unbound forms throughout z-ion series. Lower uptake in bound form was caused from protection from exchanging.
LQT G
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Uptake Bar Plots
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Uptake Bar PlotUptake Bar PlotStdStd DevDev
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Acknowledgements to Acknowledgements to DynamXDynamX Team!Team!
Rose Lawler ([email protected]) Keith Fadgen and Michael Eggertson Beta Collaborators Stephane Houel, Ying Qing Yu, and Weibin Chen Asish Chakraborty, Scott Berger, and Alan Millar
and numerous others