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How to Best Utilize your QE/QE Plus
for Maximum Peptides IDs and for
Peptide Quantitation
Tara Schroeder
Josh Nicklay
Scott Peterman
Yue Xuan
Part 1: Bottom Up Proteomics
Key Elements to Bottom Up Proteomics
• Reliable HPLC
• Sensitive column
• Stable spray
• Clean samples
• Optimized instrument method
• Fast and accurate data processing software
We will focus here!
3
QE Method Editor
Import Tune File
Parameters to be optimized with nano source:
S-lens, capillary temperature, and spray voltage
Screen shot from Tune page
4
QE Method Editor
5
QE Method Editor
6
QE Method Editor
50 ms
Advanced Options:
1. In-source CID
2. Spectrum Data Type
3. Microscans
4. MSX (multiplexing)
5. Stepped NCE
7
Chromatographic Peak Width Setting
• Chromatographic peak width setting dictates how
often an AGC prescan is collected
• Example: If you have narrow peak widths it is a critical
for the instrument to take more frequent prescans
based on the dramatic changes in ion flux
8
Resolution Settings
Recommendation:
Full MS1: 70K, dd-MS2: 17.5K
Note: For tryptic digests, 17.5K resolution is
usually an adequate setting for MS2 since
the spectra are less complex and fragment
ions are mostly +1 ions
9
Resolution and Transient Length
Resolving Power
at m/z 200
Resolving Power
at m/z 400
Transient length Approximate scan
(ms)
speed (Hz)
17,500
12,500
64
13
35,000
25,000
128
7
70,000
50,000
256
3
140,000
100,000
512
1.5
280,000*
200,000
1024
<1
*Resolution setting available with Enhanced Resolution Mode on QE Plus
Important things to note…
• Higher resolution setting does not always result in better mass accuracy
• Higher resolution increases your ability to distinguish between m/z ions
• There is always a trade off between higher resolution and time
10
Example: MS2 Scan at 17.5K Resolution
VFSDEVQQQAQLSTIR
m/z: 924.9746+2 (1.7ppm)
Xcorr: 4.83
11
Proteome Discoverer Screen Shots
Fragment Ion
Mass Error (ppm)
Parallel Filling and Scanning
Total Cycle Time ~1 sec
Full Scan
70K
64 ms
HCD
1
HCD
2
HCD
3
HCD
4
HCD
5
HCD
6
HCD
7
HCD
8
HCD
9
Fill
Detection
256 ms
Represents varying fill times with a max IT of 50 ms to
match the 17.5K resolution setting for the MS2 scan
• Orbitrap is scanning while C-trap filling
• A longer fill time then the scan time is “wasted” time
12
HCD
10
Suggested Max Fill Time
Resolving Power Approximate scan Transient length Suggested Max Fill
at m/z 200
speed (Hz)
(ms)
Time (ms)
17,500
13
64
50
35,000
7
128
110
70,000
3
256
240
140,000
1.5
512
500
• Balance the max fill time with the transient scan time for the resolution
to make the most effective use of the parallel fill and detect capabilities
of the QE
13
Parallel Filling and Scanning (Raw Data)
RT: 22.374 - 22.436
Relative Abundance
100
10909
22.395
10898
22.378
10920
22.412
10931
22.429
80
60
Cycle Time
1.02 s
1.02 s
1.02 s
Full Scan
70K
40
20
0
100
10903
22.387
10905
10902
22.390
22.386
60
40
20
10896
22.375
10908
22.394
10901
10899 22.385
22.382
10932 NL: 1.88E6
22.434 Base Peak F: FTMS + p
NSI d Full ms2 MS
Hela2_60min_64ms_1pt5
Iso_5e4_1perc_30sDyn_1
10925
22.422
10914
22.405
80
10910
22.399
10924
22.421
10913
22.403
10917
22.409
10919
22.411
10912
22.402
10922
22.418
10921
22.416
10929
22.427
10927
22.424
MS2
17.5K
10930
22.428
0
22.38
22.39
22.40
22.41
Time (min)
Scan #
RT
14
NL: 2.19E8
Base Peak F: FTMS + p
NSI Full ms
[400.00-2000.00] MS
Hela2_60min_64ms_1pt5
Iso_5e4_1perc_30sDyn_1
Top 10 HCD
22.42
22.43
Target Values and Maximum Injection Time
Example Settings:
• Full MS1
• AGC Target: 1e6
• Max IT: 30 ms
• dd-MS2
• AGC Target: 5e4
• Max IT: 50 ms
15
Target Values and Maximum Injection Time
Complex Mixture
High Load >100ng
Complex Mixture
Low Load <100ng
Simple Mixture
MS1 Target Value
1e6
1e6
5e5
MS1 Max IT (ms)
50
50
50
MS2 Target Value
5e4
1e5
1e5
MS2 Max IT (ms)
50
110
110
Parameter
• Complex Mixture (High Load): Ion selected for MS2 are mid to high
abundance. Set a lower MS2 target value (5e4 or 2e4) to maximize
scan speed and increase number of MS2 scans acquired
• Complex Mixture (Low Load): Ion selected for MS2 are low intensity,
therefore increasing your MS2 target value (1e5) and max injection time
can result in higher quality spectra for low level ions
16
Top N and Isolation Window
Recommendations:
Loop Count: 10
Isolation Window: 2 Da
Tip: Set the isolation window to
1.5 Da on the QE Plus
17
NCE (Normalized Collision Energy)
Recommendation
NCE: 27
Note: Applied voltage (eV)
depends on NCE, m/z and
charge state factor
18
Peptide Match
Recommendation:
Peptide Match Preferred
19
Peptide Match
• Definition:
• Enables the QE to select ions with peptide-like isotopic distributions for Data
Dependent scanning.
• It recognizes the monoisotopic mass of an isotopic distribution by comparing
isotopic intensity ratios to typical peptide-like distributions.
• 3 Options:
• On: will only select ions for dd-MS2 if recognized as having a peptide-like
isotopic distribution
• “–”: isotopic pattern will have no affect on MS2 selection
• Preferred: ions with peptide isotopic pattern are triggered with preference to
ions without peptide match. However, after it selects all ions that have “peptide
match” it will continue to select other ions for dd-MS2
Tip: When analyzing a complex mixture, setting
peptide match to preferred relative to on will
maximize the number of MS2 scans collected
20
Tip: When analyzing a simple
mixture, consider turning
Peptide Match “off”
Intensity Threshold
• Intensity threshold is the minimum
precursor intensity to trigger a MS2
• The MS2 target value, maximum
injection time and underfill ratio ALL
determine the threshold
21
Intensity Threshold (Examples)
Recommended Settings
MS2 Target Value
5e4
1e5
1e5
5e4
Max IT (ms)
50
110
50
50
UnderFill (%)
1
1
1
10
1.0e4
1.0e4
2.0e4
1.0e5
Intensity Threshold
Complex Mixture <100ng
Complex Mixture >100ng
• Important to Note: If you have peptide match set “on” or “preferred”,
having a very low threshold is arbitrary
22
Dynamic Exclusion and Exclude Isotopes
Recommendations:
• Exclude Isotopes: ON
• Dynamic Exclusion (s):
“Specific to peak width”
Tip: In complex mixtures, increase
dynamic exclusion to reduce
repeat sampling and maximize
number of unique peptides
23
Example: Isotope Exclusion On, 30 sec Dyn Ex.
Hela2_60min_64ms_1pt5Iso_5e4_1perc_30...
2/25/2014 10:33:32 PM
RT: 48.30 - 55.49 SM: 7G
100
Relative Abundance
50
50.03
899.4587
48.70
939.9348
50.54
829.4296
0
53.12
764.0651
51.83
868.1370
100
XIC: 868.1370
Peptide 1
50
0
51.93
868.4183
100
XIC: 868.4183
Peptide 2
50
0
49
50
51
52
Time (min)
53
54
NL: 2.78E8
Base Peak F: FTMS + p NSI Full ms
[400.00-2000.00] MS
Hela2_60min_64ms_1pt5Iso_5e4_1perc_30s
Dyn_1
NL: 3.44E6
m/z= 868.1327-868.1413 F: FTMS + p NSI
Full ms [400.00-2000.00] MS
Hela2_60min_64ms_1pt5Iso_5e4_1perc_30s
Dyn_1
NL: 2.34E6
m/z= 868.4140-868.4226 F: FTMS + p NSI
Full ms [400.00-2000.00] MS
Hela2_60min_64ms_1pt5Iso_5e4_1perc_30s
Dyn_1
55
Relative Abundance
Hela2_60min_64ms_1pt5Iso_5e4_1perc_30sDyn_1 #28748-28829 RT: 51.81-51.96 AV: 10 NL: 1.07E7
T: FTMS + p NSI Full ms [400.00-2000.00]
828.1119
z=3
100
774.7285
z=3
445.1198
80
z=1
1072.8177
z=3
60
40
519.1388
z=1
20
Full MS
70K
900.4505
z=3
657.8395
z=2
604.3362
z=3
1011.2833
z=4
1161.5900
z=2
0
400
500
600
700
800
900
1000
m/z
24
1100
1200
1300
1400
1500
Example: Isotope Exclusion On, 30 sec Dyn Ex.
Hela2_60min_64ms_1pt5Iso_5e4_1perc_30...
RT: 48.30 - 55.49 SM: 7G
100
50
2/25/2014 10:33:32 PM
NL: 2.78E8
Dynamic Exclusion of +/- 10 ppm allows for ID
of
Base Peak F: FTMS + p NSI Full ms
50.54
[400.00-2000.00] MS
53.12
829.4296 Overlapping Isotope
Patterns
Hela2_60min_64ms_1pt5Iso_5e4_1perc_30s
764.0651
50.03
899.4587
48.70
939.9348
Peptide 1
Peptide 2
Relative Abundance
Relative Abundance
Relative Abundance
Dyn_1
Hela2_60min_64ms_1pt5Iso_5e4_1perc_30sDyn_1 #28747-28838 RT: 51.81-51.96 AV: 10 NL: 2.30E6
NL: 3.44E6
51.83
T: FTMS + p NSI Full ms [400.00-2000.00]
868.1370
m/z= 868.1327-868.1413 F: FTMS + p NSI
868.1370
100
Fullfor
ms [400.00-2000.00]
MS
Selected
MS2
z=3
100
Hela2_60min_64ms_1pt5Iso_5e4_1perc_30s
50
Dyn_1
0
90
IsotopesNL:Excluded
2.34E6
51.93
868.4183
868.4183 868.4711
m/z= 868.4140-868.4226 F: FTMS + p NSI
100
80
867.8027
z=3
Full ms [400.00-2000.00] MS
z=3
z=3
Hela2_60min_64ms_1pt5Iso_5e4_1perc_30s
50
70
Dyn_1
868.7526
0
z=3
49
51
52
53
54
55
60 50
Time (min)
868.8046
868.0837
50
Hela2_60min_64ms_1pt5Iso_5e4_1perc_30sDyn_1
#28748-28829
RT: 51.81-51.96 AV: 10 NL: 1.07E7
z=3
z=3
T: FTMS + p NSI Full ms [400.00-2000.00]
869.0861
40
828.1119
z=3
z=3
100
30
774.7285
z=3
869.1383 869.4204
445.1198
20
80
z=3
z=1
z=3
1072.8177
0
Full MS
70K
z=3
10
60
0
40
519.1388
z=1
20
657.8395
z=2
604.3362
z=3
900.4505
z=3
868.5
868.0
869.0
m/z 1011.2833
z=4
869.5
1161.5900
z=2
0
400
500
600
700
800
900
1000
m/z
25
1100
1200
1300
1400
1500
Example: Isotope Exclusion On, 30 sec Dyn Ex.
Peptide 1
Peptide 2
26
EPLGNVLFSPIcLSTSLSLAQVGAK
867.8027+3 (<1 ppm)
Xcorr: 5.51
GNFTLPEVAEcFDEITYVELQK
867.8037+3 (<1 ppm)
Xcorr: 5.09
Quick Guide to a Few Scenarios
1. I want to increase the number of MS2 scans identified
•
Click through your raw data and evaluate if you have high quality of HCD
scans. Are you seeing rich spectra?
•
Are you reaching maximum injection on most MS2 scans? If YES, increase
your MS2 max IT. If NO, increase your target value
•
Also, try increasing your threshold by increasing your underfill ratio
2. I want to increase the number of MS2 scans acquired
•
If you are analyzing a complex mixture, reduce the MS2 target value to 2e4.
You will reach the target value faster, shorten cycle time and therefore
increase the number of MS2 scan acquired
3. I want to increase the number of “unique” peptide identifications
27
•
In your search results, evaluate the “# of PSMs” per identified peptide
•
If it’s high (i.e. avg >3), increase your dynamic exclusion time to decrease
repeat sampling
•
TIP: divide “peptide groups/PSMs” to give you a percent uniqueness
Part 2: Peptide Quantitation
Define the Experiment
• What are your goals?
• Are you trying to quantitate 1000 peptides?
• Is absolute quantitation of 5 targets your priority?
• There will always be a sacrifice within the targeted experiment
• If you want extreme breadth, then you will sacrifice depth
• If you want extreme depth, then you will need to decrease the number of
your targets
29
Keys to a Successful Quantitation Experiment
• Reproducibility!!
• Reproducibility in retention time
• Reproducibility in overall signal within replicates
• Obtaining adequate scans across the peak
• This is critical for accurate quantitation
• Tip: 10 scans across the peak for a middle level target will result in
enough scans across the peak at your limit of detection
• Sample complexity
• Interferences will dictate your limit of detection
30
Questions Before Starting Analysis
• How many targets do you have?
• How wide are your chromatographic peaks?
• What is the complexity of your sample?
The answers to these simple questions will greatly
impact the design of your targeted instrument method
31
Always Think About Total Cycle Time!
Resolving Power Approximate scan Transient length Suggested Max Fill
at m/z 200
speed (Hz)
(ms)
Time (ms)
32
17,500
13
64
50
35,000
7
128
110
70,000
3
256
240
140,000
1.5
512
500
Always Think About Total Cycle Time!
Total Cycle Time ~1 sec
HCD
1
HCD
2
HCD
3
HCD
4
HCD
5
HCD
6
HCD
7
HCD
8
Fill
Detection
128 ms
Represents varying fill times with a max IT of 110 ms
to match the selected 35K resolution setting
• Total Cycle time is the length of time it takes to cycle through your entire target list
• This determines how many scans across the peak are obtained
• Sufficient amount of data points defines the peak to increase quantitative
reproducibility
33
Timed or Untimed?
• How many targets do you have?
• 10 or less: Can be run untimed
• 10 or more: Most likely needs to be timed
• How wide are your chromatographic peaks?
• Example: 20 sec wide peaks
• Want 10 scans across peak. Total cycle time should not exceed 2 sec.
• For tMS2 method, using 35K (~8Hz), target at most 16 ions at a given time.
• For SIM method, using 140K (~2Hz), target at most 4 ions at a given time
• Example: 10 sec wide peaks
• Want 10 scans across peak. Total cycle time should not exceed 1 sec.
• For tMS2 method, using 35K (~8Hz), target at most 8 ions at a given time
• For SIM method, using 140K (~2Hz), target at most 2 ions at a given time
34
Different Scan Mode Options
• Quantitation by…
• Full MS
• SIM (Single Ion Monitoring)
• tMS2 (Targeted MS2)
• All Scan modes utilize high resolution/accurate mass (HR/AM)
• Extract ions with narrow mass window (<5ppm)
• 2 Major questions that will dictate what is the best scan mode
for your experiment…
• Is your priority depth or breadth?
• How complex is your sample?
35
Global Quantitation: Full MS
• Priority: Breadth (Very high number of targets)
• Complexity: High charge density (AGC 1e6 to 3e6)
• High resolution across dynamic range (140K)
• Option of acquiring confirmatory MS2
• Qualitative Attributes: RT, accurate mass, and isotopic distribution
• Quantitative Attribute: Peak areas of precursor isotopes
36
Absolute Quantitation: SIM or tMS2?
• Priority: Sensitivity!
• What is the complexity of your sample?
• Simple mixture: SIM will most likely result in highest sensitivity
• Peptide stays intact, achieve intense signal
• Complex mixture: tMS2 is usually the best choice.
• When targeting low level ions, SIM experiments in a complex mixture is likely
limited by interferences from the matrix
• Problem: Co-eluting ion in your isolation window
• Symptom: Trap will fill faster with the interference relative to your target
• Result: Decreased sensitivity of your target
• tMS2 increases selectivity and overall improves sensitivity in the presence of
co-eluting ions
− Quantitation of fragment ions is more selective!
37
Scan Mode: SIM
• Use the highest resolution setting to resolve your target from
co-eluting species in the isolation window
• Select number of isotopes for quantitation post acquisition
• Option of multiplexing (MSX) to maximize cycle time
• Qualitative Attributes: RT, accurate mass, and isotopic distribution
• Quantitative Attribute: Peak areas of precursor isotopes
38
SIM Method
39
SIM Method
• These parameters are a good starting point!
• Highest resolution setting (140K), balanced
Max IT (500 ms), 1e5 AGC Target
• Note: 10 targets are “timed” with ≤ 2
peptides/ time segment. At 140K resolution,
that is ~2 scans/sec
Tip: Reaching max IT is your goal!
40
MSX Functionality and Parallel C-Trap Filling
Orbitrap FTMS Acquisition
AGC
AGC
Standard Operation
Orbitrap FTMS Acquisition
Inject to
C-Trap
Inject to
C-Trap
Orbitrap FTMS Acquisition
Inject to Inject to Inject to
C-Trap
C-Trap
C-Trap
AGC
AGC
Spectrum Multiplexing
Orbitrap FTMS Acquisition
Inject to Inject to Inject to
C-Trap
C-Trap
C-Trap
Multiplexing Maximizes the Cycle Time of the Q Exactive
41
MSX Functionality and Parallel C-Trap Filling
Things to consider…
• When multiplexing, the AGC setting and max IT is for each fill event.
• Higher order multiplexing require lower max IT
• (max IT)/(multiplex count) = per target max IT
• Target Value Recommendations for SIM (140K Resolution)…
• Targeting without multiplexing (Target Value: 1e5-2e5)
• Multiplexing 2-5 Targets (Target Value: 1e5)
• Multiplexing 5-10 Targets: (Not recommended due to fill time/scan time relationship)
Orbitrap FTMS Acquisition
Inject to Inject to Inject to
C-Trap
C-Trap
C-Trap
AGC
AGC
Spectrum Multiplexing
Orbitrap FTMS Acquisition
Inject to Inject to Inject to
C-Trap
C-Trap
C-Trap
Multiplexing Maximizes the Cycle Time of the Q Exactive
42
tMS2
• Generates high resolution full mass range MS2 spectra
• Parallel reaction monitoring (PRM)
• Flexibility to choose the specific fragment ions for
quantitation post acquisition
• Since it is a high resolution scan, you can extract your ion
with a narrow ppm mass window achieving high selectivity
• Qualitative Attribute: RT, fragment accurate mass, and fragment ion ratios
• Quantitative Attribute: Peak areas of selected fragment ions
43
tMS2 Method
Note: The charge state in the table
will affect the applied eV and the
high end of the scan range
44
tMS2 Method
• These parameters are a good starting point!
• 35K resolution with balanced Max IT (110
ms), 1e5 AGC Target
• Note: 10 targets are “untimed”. At 35K
resolution, 1.3s cycle time will generate ~10
scans across a 10 sec FWHM peak
45
Example: Qual/Quan Workflow
dd-MS2 Screen
Relative Abundance
RT: 0.7413 - 7.3979
100
80
Basepeak
• Identified peptide list from
search engine results
60
• Select protein of interest
40
20
• Import list (library) into
PinPointTM Software
0
1
2
4
5
Time (min)
6
tMS2
723.3663
80
Relative Abundance
7
375.6712
100
90
3
70
60
419.6975
50
PinPointTM
• Build your target list and
import in QE method
40
30
666.3446
20
• Select peptides of interest
that are both sensitive and
selective
• This process is very
streamline within PinPoint!
10
0
200
400
600
800
m/z
http://portal.thermo-brims.com/
46
Example: tMS2
C:\Thermo\...\QE_5ulmin_PlasmaCurve_4_2
Acclaim PepMap 300umx5cm, 5ul/min
RT: 0.0000 - 18.1642 SM: 5G
9/25/2013 5:28:02 AM
NL: 1.82E5
m/z=
551.3171-551.3205+
666.3437-666.3477+723.3650-723.3694
F: FTMS + p NSI Full ms2
[email protected] [150.00-875.00] MS
Genesis QE_5ulmin_PlasmaCurve_4_2
RT: 6.7261
AA: 1292684
100
Relative Abundance
AD 25amol/ul, Trainer 1fmol/ul, Plasma 50ng/ul
IGDYAGIK
Extracted Ion Chromatogram:
551.3188, 666.3457, 723.3672
(6 ppm window)
80
60
40
20
0
0
2
4
6
8
10
Time (min)
12
14
16
18
Relative Abundance
QE_5ulmin_PlasmaCurve_4_2 #1322 RT: 6.71 AV: 1 NL: 2.26E5
F: FTMS + p NSI Full ms2 [email protected] [150.00-875.00]
375.6709
z=?
100
80
tMS2:
418.7292
40
0
150
47
723.3656
z=1
397.6841
z=?
60
20
y7
185.1647
z=?
200
258.1444 291.0884
z=?
z=?
250
300
366.6656
z=?
350
551.3176
z=1
419.6972
z=?
448.5667
z=3
400
450
m/z
y6
y5
519.5727 551.3183
599.3749
z=3
z=1
z=1
500
550
600
705.8019
z=2
666.3445
z=1
650
700
736.8602
z=?
750
PinPoint: Detailed Data Analysis
48
PinPoint: Detailed Data Analysis
R2=0.999
49
PinPoint: Detailed Data Analysis
50
Guide to “How to Design an
Optimized Quan Method?” and
Troubleshooting Tips
Method Selection Flowchart
Background Complexity
Low
High
SIM
MS2
Peak Width
Peak Width
20s
5s
5s
10s
10s
Max # of Simultaneous Scan Events
1 target @ 140k
2 targets @ 70k
2 target @ 140k
4 targets @ 70k
4 target @ 140k
8 targets @ 70k
Max # of Simultaneous Scan Events
4 targets @ 35k
8 targets @ 17.5k
Multiplex and/or time
as needed for # of
targets
52
20s
8 targets @ 35k
16 targets @ 17.5k
16 targets @ 35k
32 targets @ 17.5k
Remember to balance
max inject time(s) to
transient lengths!
Method Optimization
Are you reaching max
IT @ apex?
No
Yes
Is your target low level?
Yes
No
Yes
Do you have ≥10 scans
across your peak?
No
53
No
Did you detect your
target?
Yes
No
Adjust
timing or
increase
msx
Did you detect your
target?
All is well!
Troubleshoot
for low level
target
Yes
Do you have ≥10 scans
across your peak?
Troubleshoot
interference
Increase AGC
if <2E5 and/or
troubleshoot
interference
All is well!
Yes
No
Decrease
max IT or
AGC if >1E5
Method Troubleshooting
• Interference in your isolation window?
• Decrease isolation width
• Switch from SIM to tMS2
• Adjust gradient conditions
• Target is low level and close to limit of detection?
• If you are reaching target value, increase target to 2e5
• If you are not reaching target, then increase max IT as high as
peak width allows
• Play close attention to the timing of your targets. If you have very
reproducible RT, then try to narrow your time segments which can
allow you to increase you max IT.
54