New Developments in DynamX 3 - Waters Corporation

©2014 Waters Corporation 1

Alex Muck, PhDHigh-Resolution System Support Group

[email protected]

New Developments in DynamX 3.0..and insights into pDRE


Prototype

HDX-MS Collaboration with Prof. John R. Engen

(Northeastern University)

CommercialProduct Launch

2005

2006

2007

2008

2009

2010

2011

2012

2013

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


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


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


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


Ease of UseRight from the Start


MS Files BrowserMS Files Browser


MS Files MS Files BrowserBrowserGlobal, Local, Fragment Global, Local, Fragment


Enhanced VisualizationCoverage Map, Heat Map, Difference Plot


Coverage Map Coverage Map –– v3.0v3.0Relative Fractional Uptake is DisplayedRelative Fractional Uptake is Displayed


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


Coverage Map OverlaysCoverage Map OverlaysMultiple ExposuresMultiple Exposures


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


Fractional Uptake Difference Fractional Uptake Difference Boxes Boxes if if Preferred Preferred for for VisualizationVisualization

Coverage map – clearly defines the point mutation


Heat Map Heat Map –– v3.0v3.0Relative Fractional Uptake is DisplayedRelative Fractional Uptake is Displayed


Heat Map Heat Map –– v3.0 v3.0 NEW Difference ModeNEW Difference Mode


Extending the Dynamic Extending the Dynamic Range in Range in HDMSeHDMSe

pDRE – Programmable Dynamic Range Enhancement in continuum mode


UPLC - MSE


Conventional UPLC separation and orthogonal ion mobility separation


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..


UPLC – HDMSE DRE

Illustration of BPI chromatogram intensity profile obtained using HDMSe DRE


Pusher Detector

pp--DRE Alternating Transmission SwitchDRE Alternating Transmission Switch

DRE lens

Gas cell

SCAN 1- High transmission


Pusher Detector


DRE lens

Gas cell

SCAN 1- Low transmission (5%)

Changing p-DRE lens duty cycle allows precise control of transmission.


?%

m/z

High Transmission

%

LowTransmission


m/z

Stitched via Software

%

m/z

Composite spectrum

Dynamic range = 2x105


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


BPI Scale EnhancementBPI Scale Enhancement

no DRE pDRE


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.


HDMS


HDMS as Visualized BeforeHDMS as Visualized Before


New HDMS VisualizationNew HDMS Visualization


ETDSite SpecificCumulative


Site Specific Deuterium IncorporationSite Specific Deuterium IncorporationNavigation OptionsNavigation Options


Resolved Deuterium IncorporationResolved Deuterium Incorporation


ETDETDState Comparison Bound State Comparison Bound vsvs UnboundUnbound


ETDETDDifference Difference -- Bound Bound vsvs UUnboundnbound


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


Uptake Bar Plots


Uptake Bar PlotUptake Bar PlotStdStd DevDev


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

New Developments in DynamX 3 - Waters Corporation

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