ms acquisition methods overview · 2014. 6. 4. · semi targeted acquisition methods 1 – etd-dda...

MS Acquisition Methods OverviewSYNAPT G2-Si HDMS and XEVO G2-S QTOF

Ni k T kNick TomczykHDX 2014

Waters WilmslowWaters Wilmslow

©2014 Waters Corporation 1

OutlineOutlineOutlineOutline

Instrument schematics Untargeted acquisition methods

– MSE

– HDMSE

S i T t d i iti th d Semi Targeted acquisition methods– ETD-DDA

HD DDA– HD-DDA Targeted Acquisition methods

– TOF-MRMTOF MRM– HD-MRM


SYNAPT G2SYNAPT G2--SSii HDMSHDMSSYNAPT G2SYNAPT G2 SSii HDMSHDMS



Source

Stepwave ion guide Quad

ollis

ion

cell

ollis

ion

cell

IMSg

Co Co

TOF



Source Stepwave ion guide Quad

ollis

ion

cell

ollis

ion

cell

IMS TOFg

Co Co


XEVO G2XEVO G2--S QTOFS QTOFXEVO G2XEVO G2 S QTOFS QTOF

TOF

Source

Stepwave ion guide Quad Collision

cell


XEVO G2XEVO G2--S QTOF and SYNAPT G2S QTOF and SYNAPT G2--SSiiXEVO G2XEVO G2 S QTOF and SYNAPT G2S QTOF and SYNAPT G2 SSii

SYNAPT G2-Si HDMS (IMS mode)


ollis

ion

cell

ollis

ion

cell

IMS TOF

( )

g

Co Co

Source Stepwave ion guide Quad Collision

cell TOF

SYNAPT G2-Si HDMS (TOF mode)&


XEVO G2-S QTOF

MSE


Untargeted Acquisition MethodsUntargeted Acquisition Methods1 1 -- MSMSEE1 1 MSMS

Sequential Low energy and High energy MS scans Accurate mass for all precursor ions in function 1 Accurate mass for all fragment ions in function 2 Post acquisition RT alignment enables association of fragments

to precursors.


ollis

ion

cell

ollis

ion

cell

IMS TOFg

Co Co

Fn1 Fn1 LE

Fn2


Fn2 HE


Sequential Low energy and High energy MS scans Accurate mass for all precursor ions in function 1 Accurate mass for all fragment ions in function 2 Post acquisition RT alignment enables association of fragments

to precursors.

Source Stepwave ion guide Quad TOFCollision cellg

Fn1 Fn1 LE

Fn2


Fn2 HE


ityIn

tens

i

Fn1 LE

RT

ensi

ty

Fn2 HE

InteHE


RT


ity ity

Inte

nsi

Fn1 LE

Inte

nsi

RT m/z

ensi

ty

Fn2 HE en

sity

InteHE

Inte


RT m/z


Component 1 Component 2ity ityity

p pIn

tens

i

Fn1 LE

Inte

nsi

Inte

nsi

m/zm/z

ensi

ty

Fn2 HE en

sity

ensi

tyIn

teHEIn

te

Inte


m/zm/z

HDMSE


Untargeted Acquisition MethodsUntargeted Acquisition Methods2 2 -- HDMSHDMSEE2 2 HDMSHDMS

Sequential Low energy and High energy HDMS scans Accurate mass and CCS for all precursor ions in function 1 Accurate mass and CCS for all fragment ions in function 2 Post acquisition RT and DT alignment enables association of

fragments to precursors.


ollis

ion

cell

ollis

ion

cell

IMS TOFg

Co Co

Fn1 Fn1 LE

Fn2


Fn2 HE


ityIn

tens

i

Fn1 LE

RT

ensi

ty

Fn2 HE

InteHE


RT


ityIn

tens

i

Fn1 LE D

T

RT m/z

ensi

ty

Fn2 HE D

T

InteHE D


RT m/z


Component 1 Component 2p p

Fn1 LE D

T

DT

m/zm/z

Fn2 HE D

T

DT

HE DD


m/zm/z


Low Energy spectrumFenpropimorph (304.26 m/z)at 3.5pg/µL in Red Pepper Matrixpg/µ pp

RT only alignment

High Energy spectrumFenpropimorph (304.26 m/z)at 3.5pg/µL in Red Pepper Matrix

RT only alignmentRT only alignment



Low Energy spectrumFenpropimorph (304.26 m/z)at 3.5pg/µL in Red Pepper Matrixpg/µ pp

RT and DT alignment

High Energy spectrumFenpropimorph (304.26 m/z)at 3.5pg/µL in Red Pepper Matrix

RT and DT alignmentRT and DT alignment


ETD-DDA


Semi targeted Acquisition MethodsSemi targeted Acquisition Methods1 1 –– ETDETD--DDADDA1 1 ETDETD DDADDA

Data Directed Analysis– Uses a Low energy survey to identify precursors of interest

o Criteria include Intensity, charge state, isotope pattern, include/exclude lists

– After processing survey instrument performs dedicated MS/MS on After processing survey, instrument performs dedicated MS/MS on each precursor meeting criteria

– After MS/MS scans complete another MS survey scan in acquired and dprocessed



Fn1 - Survey


Col

lisio

n ce

ll

Col

lisio

n ce

ll

IMS TOF

C C

Fn2 – MS/MS 1

Source Stepwave ion id Quad is

ion

ell

isio

n el

l

IMS TOF

Fn2 – MS/MS 1

Source guide QuadCol

l ce

Col

l cIMS TOF



Fn2 – MS/MS 2


ollis

ion

cell

Col

lisio

n ce

ll

IMS TOF

C C

Fn1 - Return to survey



Data Directed Analysis– Uses a Low energy survey to identify precursors of interest

o Criteria include Intensity, charge state, isotope pattern, include/exclude lists

– After processing survery instrument performs dedicated MS/MS on After processing survery, instrument performs dedicated MS/MS on each precursor meeting criteria

– After MS/MS scans complete another MS survey scan in acquired and dprocessed

In addition to CID it is possible to choose ETD as fragmentation In addition to CID it is possible to choose ETD as fragmentation mode for certain charge state ions– Typically +2 = CID only; +3 = CID & ETD; +4/5 = ETD only


Semi targeted Acquisition MethodsSemi targeted Acquisition Methods1 1 –– ETDETD--DDADDA

New gas manifold – allows for New ion source – GD with d l

1 1 –– ETDETD--DDADDA

gselection of either argon or helium into trap/transfer cells

associated inlet

New RF Twave PCB’s

New HV power supply to sequentially drive the GD source

with higher RF


Semi targeted Acquisition MethodsSemi targeted Acquisition Methods1 1 –– ETDETD--DDADDA1 1 –– ETDETD--DDADDA

ReagentReagentVapourVapour

Reagent VialReagent Vialpp

Needle at Needle at 300V300V

ESI ESI sprayspray

--300V300V

sprayspray

Discharge Discharge RegionRegion



During the interscan the front end of instrument is put into –ve ion pmode and Trap T-wave is filled with reagent ions



During scan the analyte ions are transmitted to the Trap T-wave where reaction with reagent ions where reaction with reagent ions occurs.Resultant ETD fragment ions are transferred though rest of instrument


instrument


+1

+1481.2624

+1

+2+3

472.913+3

+2



CID of 708.8+2 ion

ETD of 472.9+3 ion

ETD of 619.6+3 ion


HD-DDA


Semi targeted Acquisition MethodsSemi targeted Acquisition Methods2 2 –– HDHD--DDADDA2 2 HDHD DDADDA

HD-DDA is similar to traditional DDA in its acquisition sequence

In addition uses ion-mobility to provide – enhanced selection criteria in survey – enhanced sensitivity in MS/MS


Semi targeted Acquisition MethodsSemi targeted Acquisition Methods2 2 –– HDHD--DDA; DDA; Enhanced selectivityEnhanced selectivity2 2 HDHD DDA; DDA; Enhanced selectivityEnhanced selectivity

Additional selection criteria for HD-DDA Makes use of correlation of m/z, DT and charge state

DT

©2014 Waters Corporation 38m/z


Additional selection criteria for HD-DDA Makes use of correlation of m/z, DT and charge state

+2+3+4,5

+1

DT



Additional selection criteria for HD-DDA Makes use of correlation of m/z, DT and charge state Remove +1 charged background from survey spectrum.

+2+3+4,5

DT


Semi targeted Acquisition MethodsSemi targeted Acquisition Methods2 2 –– HDHD--DDA; DDA; Enhanced MS/MS sensitivityEnhanced MS/MS sensitivity2 2 HDHD DDA; DDA; Enhanced MS/MS sensitivityEnhanced MS/MS sensitivity

Uses the correlation of m/z and DT to increase duty cycle on fragment ions during MS/MS

Uses the principal of Target Enhancement mode in an ion-mobility experimentmobility experiment.


Semi targeted Acquisition MethodsSemi targeted Acquisition MethodsTarget and Wideband Target and Wideband EnhancmentEnhancmentTarget and Wideband Target and Wideband EnhancmentEnhancment



Pusher

Collision Cell

In normal operation the ion beam leaving the lli i ll i h th collision cell is homogenous on the

microsecond time frame



Pusher

Collision Cell


microsecond time frame Regardless of when the pusher fires the

same population of ions is sampled.same population of ions is sampled.



Pusher

Collision Cell


microsecond time frame Regardless of when the pusher fires the

same population of ions is sampled.same population of ions is sampled. The next push cannot occur until the sample

packet of ions has reached the detector This results in a duty cycle of approx 20%



Pusher

Collision Cell

Target enhancement releases ions in packets f th lli i ll t k i t lfrom the collision cell at know intervals



Pusher

Collision Cell


The released ions undergo a mini Time-of-flight to the pusher region separating based on m/z.on m/z.



Pusher

Collision Cell



By setting the time the pusher fires after a release of a packet of ions you can target a specific m/z range



Pusher

Collision Cell



By setting the time the pusher fires after a release of a packet of ions you can target a specific m/z range


This increases duty cycle for the targeted m/z to 100%


Target EnhancementSync m/z with Tof

Normal

push

Duty Cycle

Using MASS



N l d t l T t E h t


Normal duty cycleTof, HDMS mode

Target EnhancementTof mode


Pusher

Collision Cell

Fragmentation of precursors before IMS lt i ti f f t h th t results in a separation of fragments such that

they arrive in the trap separately This means that it is possible to change the

delay after release of ions from collision cell delay after release of ions from collision cell on a push by push basis



Pusher

Collision Cell

Fragmentation of precursors before IMS lt i ti f f t h th t

Push 1

results in a separation of fragments such that they arrive in the trap separately

This means that it is possible to change the delay after release of ions from collision cell delay after release of ions from collision cell on a push by push basis



Pusher

Collision Cell


Push 2





Pusher

Collision Cell


Push 3





Pusher

Collision Cell


Push 4





Pusher

Collision Cell


Push 5

Etcresults in a separation of fragments such that they arrive in the trap separately

This means that it is possible to change the delay after release of ions from collision cell

Etc…

delay after release of ions from collision cell on a push by push basis

This results in a duty cycle of approx 90% across the m/z range



Target Wideband

EnhancementSync dt withTof

Normal

gEnhancementSync m/z with Tof

push

Sync dt withTofpush

Using Duty Cycle

gCCS & MASS

Using MASS



N l d t l T t E h t Wid b d E h t


Normal duty cycleTof, HDMS mode

Target EnhancementTof mode

Wideband EnhancementHDMS mode

Semi targeted Acquisition MethodsSemi targeted Acquisition Methods2 2 –– HDHD--DDA; DDA; Enhanced MS/MS sensitivityEnhanced MS/MS sensitivity2 2 HDHD DDA; DDA; Enhanced MS/MS sensitivityEnhanced MS/MS sensitivity

Uses the correlation of m/z and DT to increase duty cycle on fragment ions during MS/MS

Uses the principal of Target Enhancement mode in an ion-mobility experimentmobility experiment.

All MS/MS spectra are acquired using Wideband Enhancement All MS/MS spectra are acquired using Wideband Enhancement resulting in – Better quality MS/MS for low abundant species– Less time being spent on each MS/MS to produce good quality

spectrum– More MS/MS acquisitions per secondMore MS/MS acquisitions per second


Effect of WBE on MSMS TICEffect of WBE on MSMS TICEffect of WBE on MSMS TICEffect of WBE on MSMS TIC

WBE ON

Data from 15th MSMS

WBE OFF


MSMS Function1st MSMS 15th MSMS

MSMS of VILAGEVTTPVTVRMSMS of VILAGEVTTPVTVRMSMS of VILAGEVTTPVTVRMSMS of VILAGEVTTPVTVR

(switched at same point in LC peak)

WBE OFF


WBE ON


MSMS of NIVVILPSSGERMSMS of NIVVILPSSGERMSMS of NIVVILPSSGERMSMS of NIVVILPSSGER


WBE OFF


WBE ON


Effect of HDC on time in 15Effect of HDC on time in 15thth MSMSMSMSEffect of HDC on time in 15Effect of HDC on time in 15 MSMSMSMS

Function 16 WBE OFF

Function 16 WBE ON


MRM


Targeted Acquisition MethodsTargeted Acquisition Methods1 1 –– MRMMRM1 1 MRMMRM


ollis

ion

cell

Col

lisio

n ce

ll

IMS TOF

TE O

N

C C T

Targeted MS/MS scan Targeted MS/MS scan Target enhancement enabled to provide

100% duty cycle on fragment ion of choice Quantitation done using accurate mass Q g

extracted ion chromatogram



Source Stepwave ion guide Quad TOF

TE O

N

Collision cell

T

Targeted MS/MS scan Targeted MS/MS scan Target enhancement enabled to provide

100% duty cycle on fragment ion of choice Quantitation done using accurate mass Q g




Full Scan MS – S+1.25 pg on column

MRM (EDC)– S+1 25 pg on column1.25 pg on column



Full Scan MS – S+250 fg on column

MRM (EDC)– S+250 fg on column250 fg on column



250fg to 12.5fgon column


HD-MRM v.1


Targeted Acquisition MethodsTargeted Acquisition Methods2 2 –– HDHD--MRM MRM –– v 1v 12 2 HDHD MRM MRM v.1v.1


ollis

ion

cell

Col

lisio

n ce

ll

IMS TOF

WBE

ON

C C W

Targeted MS/MS scan Targeted MS/MS scan Wide band enhancement enabled to provide

90% duty cycle on fragment ions Quantitation done using accurate mass Q g




Many possible transitions from compounds possible (especially for peptides)

Which one to choose for each matrix? Which one to choose for each matrix?

HD-MRM provides flexibility on transition to use and ultimately provide best possible LOD.



HDMRM (HDC) – S+500 amol on column

y3

y7y4y2y1

iPhe

y8y6y5

MSMS – S+MSMS S+500 amol on column



HDMRM (HDC) – S+527 > 839

[M+2H]+2 >y7+1

HDMRM (HDC) – S+527 > 359

[M+2H]+2 >y3+1

MSMS – S+527 > 839

MSMS – S+527 > 359

[M+2H]+2 >y3+1[M+2H]+2 >y7+1 [M+2H]+2 >y3+1

HDMRM (HDC) – S+527 > 120

[M+2H]+2 >iPhe+1

MSMS – S+


MSMS S+527 > 120

[M+2H]+2 >iPhe+1

HD-MRM v.2




ollis

ion

cell

Col

lisio

n ce

ll

IMS TOF

TE O

N

C C T

Targeted MS/MS scan Targeted MS/MS scan Separation of precursors based on CCS

(isomers, protomers, matrix interference etc) Target enhancement enabled to provide g p

100% duty cycle on fragment ion of choice Quantitation done using accurate mass



Targeted Acquisition MethodsTargeted Acquisition Methods2 2 –– HDHD--MRM MRM –– v 1 0v 1 02 2 HDHD MRM MRM v.1.0v.1.0

XIC1 component

MS1 component

IMS - 2 components

IMS/CID - 2 MS/MS spectra

A3.69 to 4.18ms

A

B4.23ms to 4 67ms

B


4.67ms

Targeted Acquisition MethodsTargeted Acquisition Methods2 2 –– HDHD--MRM MRM –– v 1 0v 1 02 2 HDHD MRM MRM v.1.0v.1.0

AM bil 528 A3.69 to 4.18ms

B

Mobilogram 528 IMS only

B4.23ms to 4.67ms

IMS/CID/TofMobilogram 528>249

MS/CID/TofXIC 528>249

A

B


What we have coveredWhat we have coveredWhat we have coveredWhat we have covered

Instrument schematics Untargeted acquisition methods

MSE – MSE

– HDMSE

Semi Targeted acquisition methods– ETD-DDA

HD DDA– HD-DDA

Targeted Acquisition methodsTargeted Acquisition methods– TOF-MRM– HD-MRM


questions / queries / comments / requests

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ms acquisition methods overview · 2014. 6. 4. · semi targeted acquisition methods 1 – etd-dda...

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