TO DOWNLOAD A COPY OF THIS POSTER, VISIT WWW.WATERS.COM/POSTERS ©2013 Waters Corporation

m/z200 300 400 500 600 700 800 900

%

0

100y''7

808.3943

y''6

711.3413Y

136.0756 y''5

610.2937

249.1593y''7 2+

404.7013

809.3963

810.3986

METHODS

Data was acquired using dedicated research builds of MS

instrument control software. The instrument was operated at

>20,000 resolution throughout.

1. Multimode Acquisition

In the multimode acquisition (shown in Figure 3.), the

instrument is configured to cycle through four acquisition modes to produce a composite MSE and HDMSE experiment.

INTRODUCTION

Hybrid quadrupole - ion mobility - TOF mass spectrometers in

combination with high performance liquid chromatography

(UPLC) are extremely versatile and well established platforms for the investigation of complex mixtures using both data

dependent (DDA) and data independent (DIA) strategies.

A schematic showing the Synapt G2-S instrument is shown in Figure 1. In Figure 2, some of the component parts of this

instrument are listed with some of their modes of operation listed underneath. This shows that, at least in principle, there

are many hundreds of possible modes of operation of such an instrument.

A subset of these can be selected to provide the maximum

benefit for each application.

In this poster some novel combinations of modes are tested

and their utility is examined in the context of a number of real world applications.

NOVEL ACQUISITION STRATEGIES FOR TOF AND IMS-TOF MASS SPECTROMETERS

Keith Richardson1, Jason Wildgoose1, Martin Green1, Stephen Platt1, Martin Palmer1, Chris Hughes1, Mark Wrona2, Asish Chakraborty2, Arkadiusz Grzyb3 1Waters Corporation, Manchester UK; 2Waters Corporation, Milford, MA; 3Inquiry Software, Bialogard, Poland

References

1. Chris Hughes et. al. MP24 ASMS 2013

2. Wildgoose et. al. ASMS 2005. A comparison of methods of improving the duty

cycle on orthogonal TOF mass analysers.

3. www.matrixscience.com

CONCLUSION

The Q-IMS-TOF is an extremely versatile platform.

It’s flexibility permits new acquisition modes to be

defined which address the needs of diverse applications. Three new acquisition types have been

introduced.

Multimode Acquisition. This combines the separation

power of ion mobility with the high dynamic range of

TOF-MS. Linearity and specificity have been demonstrated in a drug metabolism study.

Enhanced DDA. Quantitative reproducibility is shown

for a labelled peptide spiked into a digest of a

biotheraputic drug. An HD-DDA experiment allows the identification of over 1000 proteins in 500ng of

E.coli.

TOF-MRM and HD-MRM. New scheduled acquisition

modes enabling large scale targeted quantitative

experiments.

OVERVIEW

The versatility of the Q-IMS-TOF platform is discussed

Three novel acquisition modes are introduced

Quantitative and qualitative results are demonstrated

in a mixed MS/IMS-MS experiment

Results from a quantitative DDA experiment are

presented

Over 1000 proteins are identified from 500ng E.coli in

an enhanced DDA experiment

Figure 2. Some of the supported operating modes for seven selected components of the Synapt G2-S instrument. It is clear that there are

many hundreds of operating modes of the instrument as a whole. Different combinations of these modes can be selected to optimize particular applications.

Figure 5. Multimode Acquisition: Drug Metabolism Study.

Verapamil standard dilution series. Manually integrated peak areas for the precursor were obtained from the MS data (Function 1. in

Figure 3.).

The ion mobility cell is maintained at constant pressure

throughout the experiment to avoid rapid changes of gas pres-sure; the intentional loss of mobility separation in functions 1.

and 2. is achieved through a suitable choice of transfer cell travelling wave parameters.

Multimode Acquisition: Drug Metabolism Study

Verapamil was spiked into rat liver microsome matrix and was

analysed by UPLC-MS at concentrations ranging from 0.062µM to 0.25µM. Verapamil was incubated in rat liver microsomes

for 30 min’s along with controls prior to quenching with 2:1

Acetonitrile, centrifugation and direct supernatent analysis by UPLC-MS. Results in Figures 5 & 6.

2. Enhanced DDA Acquisition Modes

Figure 4. illustrates an “HD-DDA” approach intended to sepa-

rately optimise survey and MSMS conditions in a single ion mo-bility enabled experiment. In survey mode, ion mobility sepa-

ration may be used to provide additional peak capacity (alternatively the transfer cell could be used to remerge the

ion beam for enhanced dynamic range).

CID is performed in the trap and the fragments are separated by ion mobility. In “Wideband Enhancement” (WBE) mode

[1,2] the pusher is synchronised to allow maximum TOF duty cycle for each singly charged fragment. Furthermore, the TOF

may be tuned for higher sensitivity (at some cost to resolu-tion) to further enhance the ion signal for weak fragments.

Include lists of unlimited length are also supported. In addi-tion, regularly spaced survey scans may be requested in quan-

titative experiments.

Figure 3. Multimode Acquisition. This essentially consists of par-

allel MSE (Functions 1. and 2.) and HDMSE experiments (Functions 3. and 4.), simultaneously realizing the respective quantitative and

qualitative potential of the two types of experiment.

Figure 9. Enhanced DDA: Proteomics. The TIC for all 15 MSMS functions in a 120 min separation of 400ng

of the E.coli digest standard. The large increase in signal in the functions is a direct consequence of the use of WBE to maximize TOF duty cycle. One consequence of this is that the target TIC value is

reached in a single 0.1s MSMS scan for the majority of precursors resulting in a 30% increase in the total number of switches com-

pared with the same experiment with WBE disabled.

Figure 4.Schematic of the HD-DDA acquisition. Separation in the ion

mobility cell (not shown) is enabled throughout the experiment.

Enhanced Quantitative DDA: Biotheraputic Drug

A 13C labeled reference version of the peptide TYPTNGYTR was spiked into a trypic digest of an antibody, Trastuzamab at ap-

proximately 10% and 1% relative to the native peptide. In each case, seven replicate quantitative DDA experiments were

performed in which survey scans were requested at regular 0.8s regular intervals. Results in Figure 7.

Enhanced DDA: Proteomics

400ng and 500ng of a cytosolic E. coli tryptic digest standard

was injected onto a nanoACQUITY system (Waters Corpora-tion), equipped with a C18 20 mm x 180 µm trap column and

an C18 25 cm x 75 µm analytical reversed phase column. In all

cases the gradient length was 120 minutes.

The survey scan time was 0.2s, the MSMS scan time was 0.1s and a maximum of 15 components selected for MSMS in each

survey scan. The switching criteria were: MS to MSMS on in-tensity threshold 7000/s, and MSMS to MS on integrated TIC

threshold 100,000 or maximum 0.2s. WBE was employed dur-ing the MSMS experiments. Results in Figures 8 & 9.

Figure 6. Multimode Acquisition: Drug Metabolism Study.

Rat liver microsomes after t=30 min’s incubation with Verapamil. A. High energy MS data (Function 3.), B. High energy HDMS data

(Function 4.) at drift time 1.8 ms demonstrating the extra specificity afforded by ion mobility separation.

Figure 1. Schematic of the Synapt G2-S HDMS instrument.

RESULTS

The authors would like to thank Dr.’s Joanne Connolly

and Hans Vissers for their assistance and input.

3. TOF MRM and HD-MRM Modes

In addition, two new scheduled MSMS acquisition modes are

available. These modes are described elsewhere [1]. An

unlimited number of precursors may be monitored, up to 32 compounds simultaneously. Again, pusher synchronization

(WBE) is used to optimize duty cycle in MS and IMS experi-ments. Alternatively, IMS may be used to obtain extra speci-

ficity not available in traditional MRM experiments.

A: No IMS

B: IMS

Figure 8. Enhanced DDA: Proteomics.

Protein identifications from 500ng of the E.coli digest standard ob-tained using the enhaced HD-DDA and TOF-DDA methods as well as

an HDMSE experiment. All data was processed using PLGS v3. HDMSE data was searched in PLGS, while DDA data was searched using Mascot Server. While there is considerable overlap between

the methods, both the HDMSE and HD-DDA methods return a signifi-cant number of unique proteins. The spectrum identification rate in

HD-DDA was 22% for 25k switches.

DISCUSSION

The multimode data demonstrates that it is possible to hybrid-

ize the well established MSE and HDMSE techniques. More analysis is required to reveal the full potential of this data, but

the addition of non-mobility separated spectra to an HDMSE experiment is a promising approach to extending linear dy-

namic range in complex matrices.

For some applications, high energy MS data may not be re-quired (allowing a three function experiment), and the relative

scan times could be further optimized.

The use of regularly spaced survey spectra in a DDA experi-ment has enabled highly reproducible quantitative results to be

obtained over a wide in-spectrum dynamic range.

The significant increase in the quality of results obtained with

HD-DDA is largely a result of the extra sensitivity enabled by ion mobility separation of fragment ions along with pusher

synchronization (WBE). Also important is the ability to end ac-quisition of MSMS data when the integrated TIC for the frag-

ments of a given precursor reaches a target value. This allows higher duty cycle to be allocated to remaining precursors at a

given retention time.

Since the MSE and HDMSE results are to some extent comple-mentary, analysis of the differences might yield improvements

to both or even suggest new hybrid approaches.

As well as further optimizing the acquisition methods described above, future work will involve the development of enhanced

informatics tools to facilitate deeper interpretation of the data.

Figure 7. Enhanced Quantitative DDA: Biotheraputic Drug

MSMS of TYPTNGYTR taken from a quantitative DDA experiment. The spiked 13C labeled version of the peptide was identified at a rela-

tive concentration of 9.8 ± 0.1% and 0.82 ± 0.01% over seven rep-licate injections.

NO CID

TRANSFER

REMERGE

REMERGE

NO CID

CID

MAINTAINSEPARATION

MAINTAINSEPARATION

CID

TOFMODE

TOFMODE

IMSMODE

IMSMODE

DATASYSTEM

IONMOBILITY

SEPARATING

SEPARATING

SEPARATING

SEPARATING

1

3

2

4

Fn.

0.00E+00

2.00E+05

4.00E+05

6.00E+05

8.00E+05

1.00E+06

1.20E+06

1.40E+06

1.60E+06

1.80E+06

0 0.05 0.1 0.15 0.2 0.25 0.3

Re

spo

nse

Verapamil Concentration ng/ul

1027 Proteins Identified in 500ng E.coli.

606

30

244

54

125

29

123

HD DDA[1027]

HDMSE

[883]

TOF DDA[719]

0.00E+00

2.00E+08

4.00E+08

6.00E+08

8.00E+08

1.00E+09

1.20E+09

1.40E+09

1.60E+09

1 3 5 7 9 11 13 15

TIC

MSMS Function Number

WIDEBAND ENABLED: 22407 Switches

WIDEBAND DISABLED: 17541 Switches