Achieving Peak Performance with

Q Exactive Instrument Software 2.2 SP1

Thermo Fisher Scientific, Bremen, Germany

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1

New Instrument Control SW 2.2 - Overview

General
AGC improvements for UHPLC
Exact Mass Calculator
Centroid mode
Intelligent beam management

Tune
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
2

General Improvements
1. AGC Improvements for Rapid Chromatography
2. Exact Mass Calculator
3. Data Acquisition: Centroid Mode
4. Intelligent Beam Management

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3

1. AGC Improvements

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4

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

Example: AGC (IC-SW 2.1)

In some cases of fast changes of the ion flux (high concentration & UHPLC)
100
90

80

Relative Abundance

70
60
50
40
30
20
10
0

100
90

Relative Abundance

80
70

60
50
40
30
20
10

~ 15%Diff area (A vs B)

0
2.80

2.85

2.90

2.95
Time (min)

3.00

3.05

3.10

New AGC Concepts: What is Different for the User?

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-toScan Mode

Relative Abundance

RT: 2.4 - 2.6

100

triplicates

80
60
40
20

Relative Abundance

0
100
80
60
40
20

Relative Abundance

0
100
80
60
40

Much better reproducibility and

mapping of the peak (~ 3% Diff)

20
0
2.40

2.45
Time (min)

2.50

2.55

2. Exact Mass Calculator

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9

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
Main requirements:

automated solution based on elemental composition

im-/export functionality from/to Excel

Availability in Tune and Method Editor

10

Exact Mass Calculator

TUNE

11

Method Editor

Exact Mass Calculator

Lists can be exported to or imported from Excel:

12

3. Centroid mode

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13

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

14

Centroid Mode in the Method Setup

15

Availability Through Method Editor

16

Availability Through Tune

Instrument Status Tree
Advanced user Role

right-click

17

180000

8000

160000

7000

140000
120000
100000

Centroid

80000

Profile

60000
40000

Data Volume [kB]

Data Volume [kB]

Data Volume Reduction

6000
5000
Profile
3000
2000

20000

1000

0
FS 17k

FS 35k

FS 70k

Centroid

4000

SIM 17k

SIM 35k

SIM 70k

This data is from direct infusion of calmix for 5 min by using a FS and a tSIM
experiment.
As a rule of thumb, data volume is reduced by a factor of 3 for FS
experiments.

18

Where is This Data Volume Reduction Coming From?

In-spectrum stick plot:
7 data points / peak

In-spectrum point-to-point plot

(default view)

In-spectrum stick plot

(default view):
1 data point / peak

19

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

20

4. Intelligent Beam Management

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21

Intelligent Ion Beam Management

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

HCD cell

C-Trap

Quadrupole
Mass Filter

S-lens
Ion Source

When there are large amounts of matrix

material injected into the mass
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.
22

Orbitrap
Mass Analyzer

Intelligent Ion Beam Management

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

HCD cell

C-Trap

Quadrupole
Mass Filter

S-lens
Ion Source

Becuase the Q-Exactive is a pulsed

device, once the C-Trap is filled and
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.

23

Orbitrap
Mass Analyzer

Early Software Constant Filter Mode

265 ms

Full MS

64 ms

HCD

HCD

HCD

HCD

HCD

HCD

HCD

HCD

HCD

HCD

10

Full MS

split lens:
open according
to injection times

Quad

Quad operating
in isolation mode

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.
24

New in SW 2.2: RF-only Mode

Full MS

HCD

HCD

HCD

HCD

HCD

HCD

HCD

HCD

HCD

HCD

10

Full MS

split lens:
open according
to injection times

Quad operating
in RF-only mode

Quad operating
in isolation mode

Quad in fullscan =
wide isolation range

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
25

New in SW 2.2: RF-only Mode

Full MS

HCD

HCD

HCD

HCD

HCD

HCD

HCD

HCD

HCD

HCD

10

Full MS

split lens:
open according
to injection times

Quad operating
in RF-only mode

Quad operating
in isolation mode

Quad in fullscan =
wide isolation range

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
26

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

Protein Groups
Title
Number ofAxisProtein
Groups

3500

3000

5e4

1e5

1e6

50000

100000

1000000

3082

3138

3089

2500

2000

1500

1000

500

0
Protein Groups

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.
27

Intelligent Ion Beam Management

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

HCD cell

C-Trap

Quadrupole
Mass Filter

S-lens
Ion Source

Orbitrap
Mass Analyzer

The new software now includes a test

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.

28

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
Calibration solution takes
16mins
Stay spray TIC variation < 10%
Fresh clean Calibration solution

29

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
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!

30

Changes in the Tune Window

1. Calibration Reports
2. Direct LC Control
3. Auto Source Settings

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31

1. Calibration Reports

Direct link to pdf document

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

32

Mass Calibration Report

Calibration
History
Header

Calibration
Values

Calibration
Plot

33

2. LC Direct Control from Tune

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

34

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.

Tune Instrument Direct Control

35

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:

Proxeon EASY-nLC 1000
Dionex Ultimate 3000 systems

Third party instruments are not supported yet

36

3. Source Gases - Autodefaults

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

37

Example: HESI Default Settings

Default settings available for APCI as well.

Default values can be looked up in the online help.

38

Data Independent Analysis

1. Data Independent Acquisition (DIA)
2. MSX-DIA

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39

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.

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).

40

Targeted MS/MS Classic Approach for Quantification

684.340

100

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

90
80

Relative Abundance

70
60
50

488.279

40

541.275
599.837

30

760.329
786.879

470.735

20
10

877.897

640.797

360.705

945.397
1001.935 1105.478

0
300

400

500

600

700

800
m/z

41

900

1000

1100

1200

AIF All-in Approach for Quantification

684.340

100

Covers entire mass range

Fast duty cycle
Quan-Query for every compound
in mass range
 limited specificity and selectivity
 limited dynamic range

90
80

Relative Abundance

70
60
50

488.279

40

541.275
599.837

30

760.329
786.879

470.735

20
10

877.897

640.797

360.705

945.397
1001.935 1105.478

0
300

400

500

600

700

800
m/z

42

900

1000

1100

1200

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
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.

43

DIA Exhaustive Approach for Quantification

684.340

100

covers entire mass range

Quan-Query for every
compounds within mass range
 less specific
 Medium dynamic range
 Low duty cycle

90
80

Relative Abundance

70
60
50

488.279

40

541.275
599.837

30

760.329
786.879

470.735

20
10

877.897

640.797

360.705

945.397
1001.935 1105.478

0
300

400

500

600

700

800
m/z

44

900

1000

1100

1200

MSX-DIA New Approach to Combine the Advantages*

684.340

100
90

highly specific
covers entire mass range
Quan-Query for every
compounds within mass range
deconvolution
 dyn. range limited by inj. time
 low
duty cycle (2,5-4 sec)
877.897

80

Relative Abundance

70
60
50

488.279

40

640.797
541.275
599.837

30

786.879

470.735

20
10

760.329

360.705

945.397
1001.935 1105.478

0
300

400

500

600

700

800

900

1000

1100

1200

m/z
*Poster: ThP26 ASMS 2012
Jarrett Egertson1; Andreas Kuehn2; Gennifer Merrihew1; Nicholas Bateman3; Brendan Maclean1; Jesse D. Canterbury4; Markus Kellmann2; Vlad Zabrouskov4;

45 Christine Wu 3; Michael J. Maccoss1

DIA and msxDIA Workflow on Q Exactive

HR/AM DIA Method
Instrument Control
Software 2.2

MS/MS Spectral
Library
Development
Using Pinpoint
DIA

Protein
Digest

Multiplexed 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.
46

Targeted Quan
Data Extraction
Using Pinpoint

Setup DIA in ME

47

MSX-DIA Setup

48

Appendix

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49

Multiplexed DIA (Example Mass Range 500-900, iso. 4Da,

Multiplexing Count 5)
100 4 m/z-wide windows = 400 m/z

500

m/z

900

Scan 1

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
50

Multiplexed DIA (Example Mass Range 500-900, iso. 4Da,

Multiplexing Count 5)
100 4 m/z-wide windows = 400 m/z

500
Scan 1

51

m/z

900

Multiplexed DIA (Example Mass Range 500-900, iso. 4Da,

Multiplexing Count 5)
100 4 m/z-wide windows = 400 m/z

500
Scan 1
Scan 2

52

m/z

900

Multiplexed DIA (Example Mass Range 500-900, iso. 4Da,

Multiplexing Count 5)
100 4 m/z-wide windows = 400 m/z

500
Scan 1
Scan 2

53

m/z

900

Multiplexed DIA (Example Mass Range 500-900, iso. 4Da,

Multiplexing Count 5)
100 4 m/z-wide windows = 400 m/z

500
Scan 1
Scan 2
Scan 3

54

m/z

900

Multiplexed DIA (Example Mass Range 500-900, iso. 4Da,

Multiplexing Count 5)
100 4 m/z-wide windows = 400 m/z

500
Scan 1
Scan 2
Scan 3
. . .
Scan 20

55

m/z

900

Multiplexed DIA (Example Mass Range 500-900, iso. 4Da,

Multiplexing Count 5)
100 4 m/z-wide windows = 400 m/z

500
Scan 1
Scan 2
Scan 3
. . .
Scan 20

56

m/z

900

Multiplexed DIA (Example Mass Range 500-900, iso. 4Da,

Multiplexing Count 5)
100 4 m/z-wide windows = 400 m/z

500
Scan 1
Scan 2
Scan 3
. . .
Scan 20

57

m/z

900

Multiplexed DIA (Example Mass Range 500-900, iso. 4Da,

Multiplexing Count 5)
100 4 m/z-wide windows = 400 m/z

500
Scan 1
Scan 2
Scan 3
. . .
Scan 20
Scan 21

58

m/z

900

Multiplexed DIA (Example Mass Range 500-900, iso. 4Da,

Multiplexing Count 5)
100 4 m/z-wide windows = 400 m/z

500
Scan 1
Scan 2
Scan 3
. . .
Scan 20
Scan 21

59

m/z

900

Multiplexed DIA (Example Mass Range 500-900, iso. 4Da,

Multiplexing Count 5)
100 4 m/z-wide windows = 400 m/z

500
Scan 1
Scan 2
Scan 3
. . .
Scan 20
Scan 21

60

m/z

900

Multiplexed DIA (Example Mass Range 500-900, iso. 4Da,

Multiplexing Count 5)
100 4 m/z-wide windows = 400 m/z

500
Scan 1
Scan 2
Scan 3
. . .
Scan 20
Scan 21

61

m/z

900

Intensity

Deconvolution

m/z

62

MSX DIA

Demultiplexing improves results !

63