The next step in the evolution of High Definition MS · 2011-11-22 · Algorithm development IM Calibration CCS calculator Apex peak detection ... —DriftScope Mobility Environment

©2011 Waters Corporation 1

Nordic MS User’s Meeting 8th November 2011 Dr Alistair Wallace Senior Product Manager Waters, Manchester, UK

The next step in the evolution of High Definition MS


Changing Goals and Challenges of MS

Conformation

Characterisation

Localisation

Identification

Quantification


Changing Goals and Challenges of MS

Conformation

Characterisation

Localisation

Identification

Quantification

Expertise

Low concentrations

Time Pressures

Complex mixtures

Complex molecules


SYNAPT G2-S Geometry






Purposeful Innovation in Perfect Harmony

G2-S

Sensitivity StepWave

Selectivity QuanTof Triwave

Speed FastDDA

HDMSE Informatics Engineered SimplicityTM


Time1.80 1.90 2.00 2.10 2.20 2.30

%

0

Xevo TQ-S

Xevo TQ

Time1.80 1.90 2.00 2.10 2.20 2.30

%

1

Rel

ativ

e io

n ab

unda

nce

ESI Positive Ion Sensitivity –Xevo TQ-S

Reserpine (50fg) UPLC/MRM, ESI +

800μL/min

>25X increase in peak area >5X increase in signal:noise


ESI Positive Ion Sensitivity – SYNAPT G2-S

SYNAPT G2-S vs SYNAPT G2

SYNAPT G2-S

SYNAPT G2


Increasing Sensitivity ...Ion Block Design

Sampling cone aperture — Increased to 0.8mm diameter

o Approx 5x increase in gas flow

— No supplemental pumping — All ions (and gas) enter

conjoined guide


High pressure ion guide design

Increase ion flux into device (more gas flow) Improve ion confinement efficiency Boundary between pumped regions causes expansion of gas and

ions o Potential means of ion loss

Less Ions + Much Less Gas

PUMP

Ions + Gas

Differential aperture RF Ion Guide


Ions + Gas

High pressure ion guide design Use larger inside diameter ion guide to capture ions

Problems — Ions confined in a large volume

o unsuitable for transit through next differential aperture — Higher gas flows

o more neutral contaminants enter system

Required RF-Ion Guide design — confine a diffuse ion cloud then concentrate for transport through differential aperture — robust

To Analyser


Bahr, Gerlich and Teloy in 19691 and onward2 — ion trapping device for studying ion-molecule reactions

An RF-Only Stacked Ring Device

Ion Entry

Ion Exit

RF (-)

RF (+)

1. Bahr R, Gerlich D, Teloy E. Verhandl. DPG (VI) 1969; 5: 131 2. Gerlich D. in State-Selected and State-to-State Ion-Molecule Reaction

Dynamics, Part 1: Experiment, Wiley: 1992

Stacked Ring ∝ e-r

Quadrupole ∝ r2


Ring Electrodes Opposite phase of RF applied to adjacent plates

Elec

tric

Fie

ld

Diffuse Ion Cloud

Ions In

Ions Out

Compact Ion Cloud

15mm

5mm

~11mm

~120mm

High pressure ion guide design Conjoined Ion Guide


StepWave Hardware (Conjoined + Second Ion Guide)


T-Wave

StepWave Hardware (Conjoined + Second Ion Guide)


Conjoined Ion Guide SIMION Model

No Electric Field

N2 Flow

1 mbar N2


With Electric Field (25 V between guides)

N2 Flow

E

1 mbar N2

Conjoined Ion Guide SIMION Model


Ions + Gas From API

Source

Rough Pump

Gas

Ions

Differential Aperture

Conjoined Ion Guides

Ion Trajectories

Additional design benefit : Robustness


Conjoined Ion Guide

(~2.5 mBar)

T-Wave Ion Guide

(10-3 mBar)

Rough Pumping Turbo Pump

Ion Block



Source Ion Guide Geometry Xevo TQ-S / Synapt G2-S


Assay Robustness –UPLC/MRM ESI + Xevo TQ-S

10ul loop injection 0.5pg/ul Verapamil in human plasma/ACN_4 new column

Time0.62 0.64 0.66 0.68 0.70 0.72 0.74 0.76 0.78 0.80 0.82 0.84 0.86 0.88 0.90 0.92 0.94 0.96 0.98 1.00 1.02 1.04

%

0

TC8_100723_2346 MRM of 1 Channel ES+ TIC (Verapamil)

2.66e70.83

Verapamil, 0.5pg/µL spiked into supernatant from 2:1

ACN:Plasma protein precipitation.

10µL injections ACQUITY BDH 2.1x 50

Plasma injection 1

Plasma injection 2000


5000 on column injections RSD of peak areas < 5%



QuanTof technology

QuanTof combines… — High field pusher — Dual stage reflectron — Novel ion detection system — …in an optimized, folded TOF geometry

Performance

— Resolution – over 40,000 FWHM — Mass Measurement – 1ppm RMS — Dynamic Range – up to 105 — Class-leading Sensitivity — Speed - 30 Spectra/sec

High resolution, exact mass,

quantitative performance — compatible with UPLC separations — compatible with complex mixtures — compatible with HDMS analysis


Identification …detection in complex mixtures

Intensity 6.04 x 106 Intensity 535


Quantification …LOQ, linearity and reproducibility

MS Mode s/n 1pg on column

On column LOQ (fg)

SYNAPT G2-S Resolution MSE >600 25 fg

SYNAPT G2 Resolution MSE >100 250 fg


Quantification …confirmation, identification, characterisation

MS Mode s/n 1pg on column

On column LOQ (fg)

SYNAPT G2-S Resolution MSE >600 25 fg

SYNAPT G2 Resolution MSE >100 250 fg


How can I increase information content without increasing time of analysis?

How can I increase throughput?

What if the components of interest do not separate on the basis of rt or m/z?

...INCREASE ANALYTICAL SELECTIVITY


Travelling Wave Ion Mobility …High Definition Mass Spectrometry (HDMS)

What is ion mobility? — The separation of compounds by size, shape, charge and mass

What is HDMS? — The combination of ion mobility and tandem MS


Time 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

%

0

100

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

%

0

100

para- terphenyl TB45

ortho- terphenyl AH115

Separation of Isomers and Conformers …From protein complexes to drug complexes

SYNAPT G2 40Ω/∆Ω

N2 Pressure 3.2mbar

T-Wave speed 1700m/sec 40V amplitude

SYNAPT G1 10Ω/∆Ω

N2 Pressure 1.0mbar

T-Wave speed 250m/sec 10V amplitude


The enabling technology …High Definition MS

2nd generation Triwave Increased ion mobility

resolution (over 40 Ω/ΔΩ) IMS at the limits of MS

detection Unique CID functionality

Algorithm development

IM Calibration CCS calculator Apex peak detection


Travelling Wave Ion Mobility …High Definition Mass Spectrometry (HDMS)

IMS

-9 -8 -7 -6

TOF MS LC

-5 -4 -3 -2 -1 0 1 2 3 4 n

10n seconds

Benefits of HDMS; 1. Increase selectivity, specificity and confidence

Remove interferences, improve detection limits, separate mixtures, increase peak capacity

2. Structural analysis – separate isomers and conformers, measure shape (cross-section), or enhance fragmentation studies


Identification and Quantification …increasing analytical peak capacity

Many applications are dealing with mixtures of increasing complexity

Biological matrices e.g. Plasma, faeces, bile, bacteria, yeast etc

Traditional MS/MS methods

Drift Time (bins)

HDMSE method

Component A Component B

Component A

Component B

Component A

Component B


Processed, RT ONLY Elevated Energy

Spectrum

Processed, RT ONLY Low Energy

Spectrum

Processed, RT & DT Elevated Energy

Spectrum

Processed, RT & DT Low Energy

Spectrum

IMS OFF IMS ON

HDMSE

Increased specificity very complex matrices

UPLC/IMS/MSE of ‘8 small molecule standards’ spiked into Japanese Tea


0

200

400

600

800

1000

1200

1400

1600

1800

2000

1D 2D-3 Fraction 2D-5 Fraction

Prot

eins

LC Method

MSE

HDMSE

Increasing Number of Identified Proteins with Increase in Peak Capacity

MSE

HDMSE

C. Elegans (worm) study


Identification and Quantification …increasing coverage at lower levels

S. oneidensis (bacterial) study

Observed in MSE and HDMSE

Observed in HDMSE only


Perfectly balanced, purposeful innovation

Versatility Ion sources MassLynx

UPLC ETD

Sensitivity StepWave

Selectivity QuanTof Triwave

Speed FastDDA

HDMSE Informatics Engineered SimplicityTM


Versatility


+

Localization, Identification, conformation …High Definition ImagingTM MALDI

For the first time, enabling routine implementation and use of ion mobility to maximize confidence in imaging studies

http://en.wikipedia.org/wiki/File:Vinblastine.svg


Characterisation …Electron Transfer Dissociation

High resolution, exact mass Reagent versatility and easy to maintain

m/z200 400 600 800 1000 1200 1400 1600 1800

%

0

100 1061.9

1061.4

1053.91040.91040.4z'4

440.2

c''3331.1z'1

159.0292.0 383.2

916.3915.8

z'5511.2 c''7

727.3

710.2c''6

612.2

557.5

c''8798.3

1032.4

1031.9

1062.4

1062.9

1063.4 c''111683.6

z'81396.5

z'71325.5

1063.9

c''101612.5

z'9;1511.5

c''111684.6 1831.71685.6

z'141977.8

1832.7 1978.8

Precursor at m/z 708 (3+) (EAISPPDAA(S)*AAPLR)

PTM Glycosylation, O-linked

m/z200 400 600 800 1000 1200 1400 1600 1800 2000

%

0

100 1032.1

1031.7

1009.6

z'5616.5

z'4487.4

c''3460.3z'3

374.3247.2

c''4589.4

488.4

c''6846.5

z'6731.5

732.5

802.5

c''7974.6

847.5

1032.6

2064.2

2063.2

1033.1

c''111447.8z'10

1217.8z'9

1089.7

1090.7 c''101332.8

1219.8

2047.2

1448.82018.2c''12

1576.9

1605.0 c''141818.1

2065.2

2066.2

2067.2

2087.2

Precursor at m/z 688 (3+) FQ(pS)*EEQQQTEDELQDK

PTM Phosphorylation


Characterisation …Time Aligned Parallel (TAP) fragmentation

Quad precursor selection

CID – 1º fragments

IMS of 1º fragments

CID – 2º fragments


Characterisation …Time Aligned Parallel (TAP) fragmentation

UPLC/TAP of Parent at 455.2910 m/z

RMS error = 0.6 mDa

RMS error = 2.2 mDa

RMS error = 1.6 mDa



Breakthrough Discoveries …Proven utility of HDMS across applications

>125 T-wave IMS publications References available

Core/Fundamental (14) Biological

Biological Macromolecules (60) MALDI Imaging (8)

Chemical Polymers/Biopolymers (21) NanoParticles (7)

Traditional Peptides/Proteomics (7) Small Molecules (21) […and >50 MSE references]

TAP

HDMS

CCS

CCS


Expanding Software Toolset for HDMS

General HDMSE and TAP data processing/visualisation — DriftScope Mobility Environment v2.1 — MS^E viewer v1.0

3rd party data interpretation — Lipidomics - SimLipid

Qualitative and quantitative proteomics — ProteinLynx Global SERVER

Biopharmaceutical characterisation — BiopharmaLynx

Hydrogen Deuterium eXchange — DynamX

Imaging — Waters High Definition Imaging (HDITM) MALDI


Engineered Simplicity


Engineered Simplicity


Summary

Building on unique capabilities of Q-Tof geometry — Making the most of UPLC for

complex mixture analysis — 30x sensitivity for significantly

improved LOD’s and LOQ’s Ion Mobility Spectrometry

— Significantly increases peak capacity for complex matrices

— Improved structural characterization

Flexibility/Usability — Ion source and UPLC options - ETD,

ASAP, APGC, HDX, 2DLC... — Intellistart, HDMS software...

http://en.wikipedia.org/wiki/File:Vinblastine.svg


To find out more…

www.waters.com/synaptG2S www.waters.com/mse

Documents

The next step in the evolution of High Definition MS · 2011-11-22 · Algorithm development IM Calibration CCS calculator Apex peak detection ... —DriftScope Mobility Environment