Achieving Peak Performance with Q Exactive Instrument ... ›...

Achieving Peak Performance withQ Exactive Instrument Software 2.2 SP1

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The world leader in serving science

Thermo Fisher Scientific, Bremen, Germany

New Instrument Control SW 2.2 - Overview

General• AGC improvements for UHPLC

• Exact Mass Calculator

• Centroid mode

• Intelligent beam management

Tune

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• Calibration report

• Direct LC control (Accela/Open AS)

• Auto Source settings

• Quadrupole Transmission Test

• Sweep Gas is turned on in Standby Mode

DIA• Advanced scan functions with Data Independent Analysis

General Improvements

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1. AGC Improvements for Rapid Chromatography

2. Exact Mass Calculator

3. Data Acquisition: Centroid Mode

4. Intelligent Beam Management

1. AGC Improvements

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AGC Improvements

Problem Statement:

• In fast UHPLC-MS/(MS) runs AGC was sometimes too „old“ compared to change of ion population:

� „saturation curve“ at high concentrations

� Bad precision and accuracy at high concentrations

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Example: AGC (IC-SW 2.1)

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In some cases of fast changes of the ion flux (high concentration & UHPLC)

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2.80 2.85 2.90 2.95 3.00 3.05 3.10

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~ 15%Diff area (A vs B)

New AGC Concepts: What is Different for the User?

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Upon the expected chromatographic peak width and the number of active targets taken from inclusion list information, the system is automatically choosing the AGC modes used for getting the best reference.

New AGC Results: t-HCD Alprazolam Product – MS2 Scan-to-Scan Mode

RT: 2.4 - 2.6

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Much better reproducibility and “mapping” of the peak (~ 3% Diff)

2. Exact Mass Calculator

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Exact Mass Calculator

Q Exactive is purchased and sold more into „routine“applications than in research environments.

People comming from nominal mass instrumentation often struggle with accurate mass calculation or find this tedious.

�Currently, this creates a barrier for routine adoption of HR/AM

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HR/AM

Main requirements:

� automated solution based on elemental composition

� im-/export functionality from/to Excel

� Availability in Tune and Method Editor


TUNE Method Editor

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Lists can be exported to or imported from Excel:

3. Centroid mode

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Centroid Mode

Results in very large data files

• Small molecule routine labs are used to centroids

• post-processing not an option in regulated environments

� Centroid data acquisition enabled in TUNE & ME

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Centroid Mode in the Method Setup

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Availability Through Method Editor

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Availability Through Tune

Instrument Status Tree

Advanced user Role

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“right-click”

Data Volume Reduction

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• This data is from direct infusion of calmix for 5 min by using a FS and a tSIMexperiment.

• As a rule of thumb, data volume is reduced by a factor of 3 for FS experiments.

Where is This Data Volume Reduction Coming From?

In-spectrum stick plot:7 data points / peak

In-spectrum point-to-point plot

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In-spectrum point-to-point plot(default view)

In-spectrum stick plot (default view):1 data point / peak

Why not Factor 7 Reduction?

• Raw data file size is dependent on all data which is written to the file (e.g. scan header information)

• The ratio of all this additional information is relatively high for QE data

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4. Intelligent Beam Management

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Intelligent Ion Beam Management

• The Q-Exactive, like all other Q-TOF/TRAP devices uses the quadrupole to rapdily select ions of interest for further mass selection

• When there are large amounts of matrix material injected into the mass spectrometer, the quadrupole needs to

HCD cell C-TrapQuadrupoleMass Filter

OrbitrapMass Analyzer

S-lensIon Source

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spectrometer, the quadrupole needs to filter the vast majority of these ions away.

• As part of this filtering processing, a large percentage of the filtered ions will be deposited on the rod set. This is true for ALL quadrupole designs.


• The Exactive Series 2.2SP1 software works to minimize the amount of time that the quadrupole needs to work filtering ions

• Becuase the Q-Exactive is a pulsed device, once the C-Trap is filled and waiting for injection of ions into the



S-lensIon Source

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waiting for injection of ions into the orbitrap, there is no need to continue to filter ions.

• The new software built into 2.2SP1 intellegently optimizes the filling/filtering routines to give maximum signal and minimize filtering times.

Early Software – Constant Filter Mode

Full MSHCD

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265 ms 64 ms

Full MS

split lens:open according to injection times

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Quad in fullscan =wide isolation range

• With the early software design, the quadrupole started filtering as soon as the previous scan was complete

• Filtered ions were almost always hitting the rods.

Quad operating in isolation mode

Quad

New in SW 2.2: RF-only Mode

Full MSHCD

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• Quad is operating in isolation mode only during injection times. • Once the C-Trap is filled, the quadrupole switches to RF-only. • Only during injection times excluded peptides hit the rods. In RF-only the complete

ion beam is forwarded through the quad and deffered at the split lens

Quad operating in RF-only mode


New in SW 2.2: RF-only Mode

Full MSHCD

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• NOTE: When the target value is set ultra-high, the ratio of filling to scanning time is such that the quads will be in filter mode for a very high percentage of the time.

• Do high target values really help? See next slide

Quad operating in RF-only mode


Hela Digest (1µg, 1% FDR, 75 min), Average (Triplicates)

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Protein Groups

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50000 100000 1000000

Protein Groups 3082 3138 3089

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Protein Groups 3082 3138 3089

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For many high-throughput, high load proteomics workflows, a larger target value provides

no extra IDs.

Always use the lowest target value possible. This is the easiest way to increase uptime.

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• Of course, even with the improved beam management and lower target values, there may be cases of customers running “extreme” sample conditions.

• The new software now includes a test to determine if the Q1 has become



S-lensIon Source

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to determine if the Q1 has become contaminated (see next slide).

• For these types of workflows, the Thermo Scientific service organization offers a number of very affordable options for frequent Q1 cleaning.

New Tool to Check for Q1 Charging Status

• Tune: Advance Mode

• Evaluation/Extra Evaluation/ Isolation Transmission Endurance Test

• Start evaluation with:

• Evaluating with Postive Mode

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• Evaluating with Postive Mode Calibration solution takes 16mins

• Stay spray TIC variation < 10%

• Fresh clean Calibration solution

New Tool to Check for Q1 Charging Status (cont.)

• How to read the Evaluation Result

• The evaluation result are presented as a Transmission Score

• A Transmission Score close to 1 indicates a clean Q

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• A Transmission Score less than 1 indicates contamination of the quad

• When the score is < 0.5 combined with a 20% decrease of protein IDs from a QC standard, the quad should be cleaned!

Changes in the Tune Window

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1. Calibration Reports

2. Direct LC Control

3. Auto Source Settings

1. Calibration Reports

Direct link to pdf document

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• All reports are stored automatically • The customer can choose between

• whole calibration report (includes all calibration procedures which have been updated

on a selected date)

• custom spectral mass calibration

• spectral mass calibration (pos or neg)

• list of all reports

Mass Calibration Report

Calibration History

Calibration

Header

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Calibration Values

Calibration Plot

2. LC Direct Control from Tune

• Access via „LC Direct Control“ icon or „Menu/Windows“ if an Accela LC instrument was configured

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LC Direct Control from Tune: Accela Systems

• Set toggle „Take pump under control“ for Accela Pump Direct Control. Check Status in Xcalibur Sequence Editor „Direct Control“.

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Tune Instrument Direct Control Xcalibur Sequence Editor

Direct Control for LC Systems

• Supported Thermo systems:

☺Accela Pump 1250

☺Accela Autosampler Open AS

☺CTC PAL Autosampler

• Thermo systems not supported yet for TUNE direct control:

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�Proxeon EASY-nLC 1000

�Dionex Ultimate 3000 systems

• Third party instruments are not supported yet

3. Source Gases - Autodefaults

• Default source voltage and gas settings are applied according to flow rate

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Example: HESI Default Settings

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Default settings available for APCI as well.Default values can be looked up in the online help.

Data Independent Analysis

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1. Data Independent Acquisition (DIA)

2. MSX-DIA

DIA − High Throughput Comprehensive Quantification Acquisition Methods with Qualitative Confirmation

• In DIA (data-independent acquisition) experiments, a set mass range is pre-defined that corresponds to predominant precursor m/z range for enzymatic peptides. MS/MS data are collected repeatedly until all precursor ions in the defined mass range are selected for fragmentation, yielding MS/MS spectra on all precursor ions.

• By acquiring MS/MS data for all precursor ions in each sample, DIA seeks to increase reproducibility and comprehensiveness of data collection within different samples. No detailed sample knowledge is required prior to the DIA-based analysis.

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• Data collection produces a complete record of quantitative data and a targeted data analysis strategy can be employed to mine additional analytes, retrospectively relying on MS/Ms spectral library.

• The new instrument control software 2.2 on Q Exactive offers two different DIA approaches: DIA and MSX-DIA (multiplexed DIA).

Targeted MS/MS – „Classic“ Approach for Quantification

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☺ highly specific☺ very fast� Misses most of the mass range� Quan-Query for prior defined compounds

☺ highly specific☺ very fast� Misses most of the mass range� Quan-Query for prior defined compounds

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488.279

877.897640.797

760.329599.837

786.879470.735

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360.705

1001.935 1105.478

945.397

AIF – All-in Approach for Quantification

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☺ Covers entire mass range☺ Fast duty cycle☺ Quan-Query for every compound

in mass range� limited specificity and selectivity� limited dynamic range

☺ Covers entire mass range☺ Fast duty cycle☺ Quan-Query for every compound

in mass range� limited specificity and selectivity� limited dynamic range

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760.329599.837

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541.275

360.705

1001.935 1105.478

945.397

DIA on Q Exactive

• Thermo – Data Independent Acquisition (DIA) on Q Exactive

• DIA method with wider isolation window (up to 50Da) sequentially scanning through the entire mass range

• multiplexed DIA (msxDIA) method using narrow isolation window width and multiplexing MS2 in a random fashion

• Advantages of DIA on Q Exactive

• Capability to use higher resolution set up for more accurate quantitative results by

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• Capability to use higher resolution set up for more accurate quantitative results by resolving analytes from interferences.

• Flexibility to use different combination of mass range and resolution to adapt different research needs.

• Easy methods set up with new Method Editor.

DIA – Exhaustive Approach for Quantification

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☺ covers entire mass range☺ Quan-Query for every

compounds within mass range� less specific� Medium dynamic range� Low duty cycle

☺ covers entire mass range☺ Quan-Query for every

compounds within mass range� less specific� Medium dynamic range� Low duty cycle

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488.279

877.897640.797

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786.879470.735

541.275

360.705

1001.935 1105.478

945.397

MSX-DIA– New Approach to Combine the Advantages*

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☺ highly specific☺ covers entire mass range☺ Quan-Query for every

compounds within mass range☺ deconvolution

☺ highly specific☺ covers entire mass range☺ Quan-Query for every

compounds within mass range☺ deconvolution

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488.279

877.897640.797

760.329599.837

786.879470.735

541.275

360.705

1001.935 1105.478

945.397

☺ deconvolution� dyn. range limited by inj. time� low duty cycle (2,5-4 sec)

☺ deconvolution� dyn. range limited by inj. time� low duty cycle (2,5-4 sec)

*Poster: ThP26 ASMS 2012 Jarrett Egertson1; Andreas Kuehn2; Gennifer Merrihew1; Nicholas Bateman3; Brendan Maclean1; Jesse D. Canterbury4; Markus Kellmann2; Vlad Zabrouskov4; Christine Wu 3; Michael J. Maccoss1

HR/AM DIA Method

Software 2.2

HR/AM DIA MethodInstrument Control

Software 2.2

Protein Digest

MS/MS Spectral Library

DevelopmentUsing Pinpoint

DIA and msxDIA Workflow on Q Exactive

DIA

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Targeted QuanData Extraction Using PinpointMultiplexed DIA

(msxDIA)

Predefined include lists and data processing is not part of Method Editor. This will be provided by third party software packages like Skyline.

Setup DIA in ME

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MSX-DIA Setup

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Appendix

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Scan 1

100 4 m/z-wide windows = 400 m/z

m/z500 900

Multiplexed DIA (Example Mass Range 500-900, iso. 4Da, Multiplexing Count 5)

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Poster: ThP26 ASMS 2012 Jarrett Egertson1; Andreas Kuehn2; Gennifer Merrihew1; Nicholas Bateman3; Brendan Maclean1; Jesse D. Canterbury4; Markus Kellmann2; Vlad Zabrouskov4; Christine Wu 3; Michael J. Maccoss1,1University of Washington, Seattle, WA; 2Thermo Fisher Scientific, Bremen, GERMANY; 3University of Pittsburgh, Pittsburgh, PA; 4ThermoFisher Scientific, San Jose, CA

Poster: ThP26 ASMS 2012 Jarrett Egertson1; Andreas Kuehn2; Gennifer Merrihew1; Nicholas Bateman3; Brendan Maclean1; Jesse D. Canterbury4; Markus Kellmann2; Vlad Zabrouskov4; Christine Wu 3; Michael J. Maccoss1,1University of Washington, Seattle, WA; 2Thermo Fisher Scientific, Bremen, GERMANY; 3University of Pittsburgh, Pittsburgh, PA; 4ThermoFisher Scientific, San Jose, CA

Scan 1


m/z500 900


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Scan 1


m/z500 900

Scan 2


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Scan 1


m/z500 900

Scan 2


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Scan 1


m/z500 900

Scan 2

Scan 3


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Scan 3

Scan 1


m/z500 900

Scan 2

Scan 3


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Scan 3

Scan 20

. . .

Scan 1


m/z500 900

Scan 2

Scan 3


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Scan 3

Scan 20

. . .

Scan 1


m/z500 900

Scan 2

Scan 3


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Scan 3

Scan 20

. . .

Scan 1


m/z500 900

Scan 2

Scan 3


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Scan 3

Scan 20

. . .

Scan 21

Scan 1


m/z500 900

Scan 2

Scan 3


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Scan 3

Scan 20

. . .

Scan 21

Scan 1


m/z500 900

Scan 2

Scan 3


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Scan 3

Scan 20

. . .

Scan 21


Scan 1


m/z500 900

Scan 2

Scan 3

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Scan 3

Scan 20

. . .

Scan 21

Inte

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Deconvolution

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m/z

Inte

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MSX – DIA

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Demultiplexing improves results !

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