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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
1. AGC Improvements
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
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
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.
triplicates
80
60
40
20
Relative Abundance
0
100
80
60
40
20
Relative Abundance
0
100
80
60
40
20
0
2.40
2.45
Time (min)
2.50
2.55
10
11
Method Editor
12
3. Centroid mode
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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15
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right-click
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180000
8000
160000
7000
140000
120000
100000
Centroid
80000
Profile
60000
40000
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.
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HCD cell
C-Trap
Quadrupole
Mass Filter
S-lens
Ion Source
Orbitrap
Mass Analyzer
HCD cell
C-Trap
Quadrupole
Mass Filter
S-lens
Ion Source
23
Orbitrap
Mass Analyzer
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
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
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
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
HCD cell
C-Trap
Quadrupole
Mass Filter
S-lens
Ion Source
Orbitrap
Mass Analyzer
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1. Calibration Reports
32
Calibration
Values
Calibration
Plot
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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
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
100
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
43
100
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
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;
MS/MS Spectral
Library
Development
Using Pinpoint
DIA
Protein
Digest
Multiplexed DIA
(msxDIA)
Targeted Quan
Data Extraction
Using Pinpoint
Setup DIA in ME
47
MSX-DIA Setup
48
Appendix
500
m/z
900
Scan 1
500
Scan 1
51
m/z
900
500
Scan 1
Scan 2
52
m/z
900
500
Scan 1
Scan 2
53
m/z
900
500
Scan 1
Scan 2
Scan 3
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m/z
900
500
Scan 1
Scan 2
Scan 3
. . .
Scan 20
55
m/z
900
500
Scan 1
Scan 2
Scan 3
. . .
Scan 20
56
m/z
900
500
Scan 1
Scan 2
Scan 3
. . .
Scan 20
57
m/z
900
500
Scan 1
Scan 2
Scan 3
. . .
Scan 20
Scan 21
58
m/z
900
500
Scan 1
Scan 2
Scan 3
. . .
Scan 20
Scan 21
59
m/z
900
500
Scan 1
Scan 2
Scan 3
. . .
Scan 20
Scan 21
60
m/z
900
500
Scan 1
Scan 2
Scan 3
. . .
Scan 20
Scan 21
61
m/z
900
Intensity
Deconvolution
m/z
62
MSX DIA
63