A Case Study on Customer Acquisiton and Retention on the ...
QStar Presentation 050503 · 2009. 11. 29. · Sample Preparation and Acquisiton. Orifice Plate...
Transcript of QStar Presentation 050503 · 2009. 11. 29. · Sample Preparation and Acquisiton. Orifice Plate...
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QSTAR Operation
May 3, 2005
Bob Seward
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Presentation CD Contents
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QSTAR Hardware
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10-7
torr
QSTAR Schematic
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QSTAR Schematic
Chernushevich et al, JMS 2001, 36: 849-865
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ESI Source
Curtain Plate
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ESI Source
Curtain Gas
Curtain Plate
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QSTAR Mass Ranges
Upper m/z Ranges
QSTAR 1 QSTAR 2TOFMS 12,000 12,000
MSMS 6,000 3,000
Lower m/z Ranges = 5
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With Turbo IonSpray Source
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TOF Analyzer
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Turbo Pumps
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ESI Source With Curtain Plate
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Curtain Plate Removed
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Source Removed Skimmer Facing Up
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Skimmer Removed
Ring
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Skimmer
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Q0
Source Removed: Q0
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Q0 Q1 Q2 - EnclosedTo Maintain CAD
Gas Pressure
Quadrupole Rail
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Stubbies and Q1
Decreasing Pressure
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Q2 and CAD Gas Supply
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Q2 Exit Lens
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DC Quad Lens: Directs Ion Beam Into TOF
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Nanospray
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Nanospray Source Cameras
CenterSide
XYZ Stage
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Move TipClose ToOrifice
Nanospray Tip Positioning 1: Be Sure That QSTAR Is In Standby Mode
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Center Side
Off Axis
Nanospray Tip Positioning 2Move Nanospray Tip Off Axis
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Nanospray Tip Positioning 3Move Tip Back
Center Side
Good Spraying Distance
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Sample Preparation and Acquisiton
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Orifice Plate Contamination
ContaminantBuildup Around
The Orifice
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Nanospray Guidelines To Reduce Contamination
• Pre-clean capillaries before pulling tips
• Inspect every tip under the microscope (200X)
• Always spray off axis from the orifice
• Cleanup the sample (dirty samples introduce contamination and require longer acquisition time)
• Acquire only long enough to get a good spectrum
• Put QSTAR in standby when not acquiring data (IS voltage is still applied when acquisition is stopped)
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Monitoring Ion Counts With The Display Meter
Keep Stop Rate Below
50,000: Dilute Or Cleanup Sample If Too High
Signal On Each Of The 4 Anodes Of
The MCP
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Wash The Capillary With Solvent (50%ACN)
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Dry The Capillaries Overnight Before Pulling The Nanospray Tips
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Performance Standards• Run before and after sample set acquisition
• Positive Mode– [Glu1]-Fibrinopeptide B, Human, Sigma F-3261– EGVNDNEEGFFSAR– 1pmol/µl, 50%ACN / 0.5%Formic Acid
• Negative Mode– Sulfated Disaccharide, V-LABS, Inc.– Di-6S (C 3203) or Di-4S (C 3202), m/z 458.0604 (1-)– 1pmol/µl, 30%MeOH / 0.1% NH4OH (v/v, using 30% NH4OH
stock)
• Prepare concentrated aliquots, store frozen– Dilute a fresh aliquot for each day’s use
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Sample Log Book• Name and project folder (if different from name)
• Sample names, descriptions, and concentrations (if known)
• Solvent composition
• Acquisition mode (+ or -)
• File names of all samples (including performance standards)
• Cleanup procedure (ex, ZipTip, HILIC, SEC)
• Any unusual occurrences (ex. software crash)
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Analyst QS Software
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Software Components• Analyst QS with oMALDI Server
– Instrument operation and data analysis• Service Pack 8
– Update to Analyst QS• Biotools
– Extension of Analyst QS for Protein/Peptide analysis • PepSea Server
– Database search using Biotools data• Scripts
– Relatively simple software additions for data analysis• Pro ID
– Protein/Peptide database search script• Install software in the order listed
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Analyst QS Overview
Manual Acquisition
Information Dependent Acquisition (IDA)
Data Analysis
Hardware Setup, Printing Configuration, Security
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Analyst QS Overview
Navigation Bar:Most functions accessible from toolbars and menus.Can be closed to save screen space.
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Analyst QS Overview
Configure Mode: Security Configuration
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Analyst QS Overview
Help Topics
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Creating a Project
“New Project” Icon
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Creating a Project
Type in Project Name – Press OK:D:\PE Sciex Data\Projects\My Project
Data FolderAcquisition Methods Folder
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(1) Open Project(2) Select Tune Mode
Startup: Open Project and Tune Mode
Instrument Status Indicator:Green -ReadyYellow -StandbyRed -Fault
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Make Sure That The Instrument Is In Standby Mode Before
Touching the Source!
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(3) View Queue Standby / Ready Mode
Startup: Open the Queue Manager
Queue Manager Can Be Minimized
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“Manual Tune” Icon
Startup: Open Manual Tuning Window
Ready Mode
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Startup
D:\PE Sciex Data\Projects\API Instrument\Tuning Cache
Temp Folder – Only 2 most recent files are saved – can copy and paste file to save
Wait a few seconds when starting/stopping or switching between standby/ready mode to prevent software freeze
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10-7
torr
Instrument Settings
***
**
*
*
* User Adjustable
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TOF MS Acquisition
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Set Acquisition Parameters: Source/Gas & MS Tabs
Source/Gas Tab MS Tab
Set Polarity In The Software Before
Setting “TOF Offset”On The Instrument
Panel
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Set to Nanospray and Adjust “TOF Offset” to Correct Value
There Are Separate “TOF Offset” Values
For Negative And Positive Modes
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Set Acquisition Parameters: Compound & Advanced MS Tabs
Compound Tab Advanced MS Tab
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Save The Acquisition Method
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Save a Method for Each Frequently-Used Analysis Mode
Open a new method when switching between TOFMS and MSMS:-Faster-Less chance of making errors
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Open Acquisition Method
(1) Ready Mode(2) Manual Tune Window Open
(3) Open File
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Product Ion Mode
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Set Acquisiton Parameters: Product Ion Mode
Compound Tab MS Tab
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Set Acquisiton Parameters: Resolution & Advanced MS Tabs
Advanced MS TabResolution Tab
Low Q1 Resolution:- Increased sensitivity- Transmit isotope envelope- TOF resolution unaffected
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Ion Transmission Efficiency
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400-1700
350-1700 (2 “Hops”)
80-1700 (3 “Hops”)
Max m/zMin m/z
> ~5
Quadrupole “Hops”To Cover m/z Range
Min – Max m/z
Quadrupole Transmission Windows, TOFMS (RF-Only)
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10-7
torr
QSTAR Schematic
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Ion Beamfrom Quad
Detector
“slice” of ions injected
Orthogonal Injection TOF
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X =width ofejected beam slice
L = distance traveled by ions between pulses
Duty cycle = x/L= 4% (m/z 100)= 12% (m/z 1000)= 26% (m/z 5000)
10 eV
TOF Cycle
Pulse
Duty Cycle of Orthogonal Injection TOF
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400-2000
400-2100
TOF Pulser Frequency Changes With Maximum m/z
Slower Frequency = Lower Transmission Efficiency
TOF Analyzer “Waits” Longer BetweenCycles – Ion Beam From Quad is Lost
During This Time
Min – Max m/z
Pulser Frequency Range: 3-20 KHz
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Q2 Pulsing (Enhancing)
![Page 67: QStar Presentation 050503 · 2009. 11. 29. · Sample Preparation and Acquisiton. Orifice Plate Contamination Contaminant Buildup Around ... Use DHB Matrix Only QSTAR Target Bruker](https://reader036.fdocuments.us/reader036/viewer/2022081619/60fbeefe714ce04a877142e4/html5/thumbnails/67.jpg)
Enhancing
Enhance a Single m/z
Enhance a m/z Range
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Ions are Stored in Q2, then Pulsed into the TOF
Q2 collision cell
QSTAR Pulsar: Pulsing ON
![Page 69: QStar Presentation 050503 · 2009. 11. 29. · Sample Preparation and Acquisiton. Orifice Plate Contamination Contaminant Buildup Around ... Use DHB Matrix Only QSTAR Target Bruker](https://reader036.fdocuments.us/reader036/viewer/2022081619/60fbeefe714ce04a877142e4/html5/thumbnails/69.jpg)
Q2 Exit Lens
![Page 70: QStar Presentation 050503 · 2009. 11. 29. · Sample Preparation and Acquisiton. Orifice Plate Contamination Contaminant Buildup Around ... Use DHB Matrix Only QSTAR Target Bruker](https://reader036.fdocuments.us/reader036/viewer/2022081619/60fbeefe714ce04a877142e4/html5/thumbnails/70.jpg)
Enhancing a Single m/zIRD & IRW are Optimized for that m/z
No effect on the Quadrupole Transmission Windows
![Page 71: QStar Presentation 050503 · 2009. 11. 29. · Sample Preparation and Acquisiton. Orifice Plate Contamination Contaminant Buildup Around ... Use DHB Matrix Only QSTAR Target Bruker](https://reader036.fdocuments.us/reader036/viewer/2022081619/60fbeefe714ce04a877142e4/html5/thumbnails/71.jpg)
Enhance All
More Quadrupole Transmission Windows are Generated
IRD & IRW are Optimized for Each Window
No Enhancing (70-1800)
Enhance All (70-1800)
![Page 72: QStar Presentation 050503 · 2009. 11. 29. · Sample Preparation and Acquisiton. Orifice Plate Contamination Contaminant Buildup Around ... Use DHB Matrix Only QSTAR Target Bruker](https://reader036.fdocuments.us/reader036/viewer/2022081619/60fbeefe714ce04a877142e4/html5/thumbnails/72.jpg)
Enhance All
![Page 73: QStar Presentation 050503 · 2009. 11. 29. · Sample Preparation and Acquisiton. Orifice Plate Contamination Contaminant Buildup Around ... Use DHB Matrix Only QSTAR Target Bruker](https://reader036.fdocuments.us/reader036/viewer/2022081619/60fbeefe714ce04a877142e4/html5/thumbnails/73.jpg)
Enhancing: Glu-Fibrinopeptide B
No Enhance Enhance All Enhance 785.8
![Page 74: QStar Presentation 050503 · 2009. 11. 29. · Sample Preparation and Acquisiton. Orifice Plate Contamination Contaminant Buildup Around ... Use DHB Matrix Only QSTAR Target Bruker](https://reader036.fdocuments.us/reader036/viewer/2022081619/60fbeefe714ce04a877142e4/html5/thumbnails/74.jpg)
Increasing Quadrupole Residence Time at Higher Mass
![Page 75: QStar Presentation 050503 · 2009. 11. 29. · Sample Preparation and Acquisiton. Orifice Plate Contamination Contaminant Buildup Around ... Use DHB Matrix Only QSTAR Target Bruker](https://reader036.fdocuments.us/reader036/viewer/2022081619/60fbeefe714ce04a877142e4/html5/thumbnails/75.jpg)
Changing CE For Individual Quadrupole Transmission Windows
Right-ClickNext to %Column
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MALDI Analysis
![Page 77: QStar Presentation 050503 · 2009. 11. 29. · Sample Preparation and Acquisiton. Orifice Plate Contamination Contaminant Buildup Around ... Use DHB Matrix Only QSTAR Target Bruker](https://reader036.fdocuments.us/reader036/viewer/2022081619/60fbeefe714ce04a877142e4/html5/thumbnails/77.jpg)
MALDI Target and AdapterUse DHB Matrix Only
Bruker AdapterQSTAR Target
![Page 78: QStar Presentation 050503 · 2009. 11. 29. · Sample Preparation and Acquisiton. Orifice Plate Contamination Contaminant Buildup Around ... Use DHB Matrix Only QSTAR Target Bruker](https://reader036.fdocuments.us/reader036/viewer/2022081619/60fbeefe714ce04a877142e4/html5/thumbnails/78.jpg)
MALDI Source
Laser Guide
![Page 79: QStar Presentation 050503 · 2009. 11. 29. · Sample Preparation and Acquisiton. Orifice Plate Contamination Contaminant Buildup Around ... Use DHB Matrix Only QSTAR Target Bruker](https://reader036.fdocuments.us/reader036/viewer/2022081619/60fbeefe714ce04a877142e4/html5/thumbnails/79.jpg)
Installing the MALDI Target
Target
![Page 80: QStar Presentation 050503 · 2009. 11. 29. · Sample Preparation and Acquisiton. Orifice Plate Contamination Contaminant Buildup Around ... Use DHB Matrix Only QSTAR Target Bruker](https://reader036.fdocuments.us/reader036/viewer/2022081619/60fbeefe714ce04a877142e4/html5/thumbnails/80.jpg)
Installing the MALDI Target
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oMALDI Server Software
• Controls laser and sample target
• Sample acquisition controlled by Analyst QS
• Start acquisition first – then switch laser on
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oMALDI ServerStart From Desktop Icon
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Pumping Down The MALDI Source
Turns Bright Green When Vacuum is
Ready
![Page 84: QStar Presentation 050503 · 2009. 11. 29. · Sample Preparation and Acquisiton. Orifice Plate Contamination Contaminant Buildup Around ... Use DHB Matrix Only QSTAR Target Bruker](https://reader036.fdocuments.us/reader036/viewer/2022081619/60fbeefe714ce04a877142e4/html5/thumbnails/84.jpg)
Removing The MALDI Plate
![Page 85: QStar Presentation 050503 · 2009. 11. 29. · Sample Preparation and Acquisiton. Orifice Plate Contamination Contaminant Buildup Around ... Use DHB Matrix Only QSTAR Target Bruker](https://reader036.fdocuments.us/reader036/viewer/2022081619/60fbeefe714ce04a877142e4/html5/thumbnails/85.jpg)
Laser Control
Pulse Rate: 20
Attenuator: 20,000
Laser On/Off
![Page 86: QStar Presentation 050503 · 2009. 11. 29. · Sample Preparation and Acquisiton. Orifice Plate Contamination Contaminant Buildup Around ... Use DHB Matrix Only QSTAR Target Bruker](https://reader036.fdocuments.us/reader036/viewer/2022081619/60fbeefe714ce04a877142e4/html5/thumbnails/86.jpg)
Positioning The Target and Sample
![Page 87: QStar Presentation 050503 · 2009. 11. 29. · Sample Preparation and Acquisiton. Orifice Plate Contamination Contaminant Buildup Around ... Use DHB Matrix Only QSTAR Target Bruker](https://reader036.fdocuments.us/reader036/viewer/2022081619/60fbeefe714ce04a877142e4/html5/thumbnails/87.jpg)
Positioning The Sample
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TOF Calibration
![Page 89: QStar Presentation 050503 · 2009. 11. 29. · Sample Preparation and Acquisiton. Orifice Plate Contamination Contaminant Buildup Around ... Use DHB Matrix Only QSTAR Target Bruker](https://reader036.fdocuments.us/reader036/viewer/2022081619/60fbeefe714ce04a877142e4/html5/thumbnails/89.jpg)
Select Two Peaks – Right Click – Re-Calibrate TOF
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Select Calibration File
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Select Calibration File
![Page 92: QStar Presentation 050503 · 2009. 11. 29. · Sample Preparation and Acquisiton. Orifice Plate Contamination Contaminant Buildup Around ... Use DHB Matrix Only QSTAR Target Bruker](https://reader036.fdocuments.us/reader036/viewer/2022081619/60fbeefe714ce04a877142e4/html5/thumbnails/92.jpg)
Calculate New Calibrations
![Page 93: QStar Presentation 050503 · 2009. 11. 29. · Sample Preparation and Acquisiton. Orifice Plate Contamination Contaminant Buildup Around ... Use DHB Matrix Only QSTAR Target Bruker](https://reader036.fdocuments.us/reader036/viewer/2022081619/60fbeefe714ce04a877142e4/html5/thumbnails/93.jpg)
Calculate New Calibrations
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Calibrate Spectrum
![Page 95: QStar Presentation 050503 · 2009. 11. 29. · Sample Preparation and Acquisiton. Orifice Plate Contamination Contaminant Buildup Around ... Use DHB Matrix Only QSTAR Target Bruker](https://reader036.fdocuments.us/reader036/viewer/2022081619/60fbeefe714ce04a877142e4/html5/thumbnails/95.jpg)
Calibrate Spectrum
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Apply to Entire File
For Highest Accuracy: Calibrate Instrument and Acquire Samples with the Same TOF Pulser Frequency
![Page 97: QStar Presentation 050503 · 2009. 11. 29. · Sample Preparation and Acquisiton. Orifice Plate Contamination Contaminant Buildup Around ... Use DHB Matrix Only QSTAR Target Bruker](https://reader036.fdocuments.us/reader036/viewer/2022081619/60fbeefe714ce04a877142e4/html5/thumbnails/97.jpg)
Recalibration of a Spectrum
![Page 98: QStar Presentation 050503 · 2009. 11. 29. · Sample Preparation and Acquisiton. Orifice Plate Contamination Contaminant Buildup Around ... Use DHB Matrix Only QSTAR Target Bruker](https://reader036.fdocuments.us/reader036/viewer/2022081619/60fbeefe714ce04a877142e4/html5/thumbnails/98.jpg)
Apply Calibrations to Sample
Uncheck Boxes
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Save As Recalibrated File
![Page 100: QStar Presentation 050503 · 2009. 11. 29. · Sample Preparation and Acquisiton. Orifice Plate Contamination Contaminant Buildup Around ... Use DHB Matrix Only QSTAR Target Bruker](https://reader036.fdocuments.us/reader036/viewer/2022081619/60fbeefe714ce04a877142e4/html5/thumbnails/100.jpg)
Precursor Ion Scanning• Q1 scans a m/z range
• Collision in Q2
• Characteristic fragment ions detected in TOF
• Output spectrum indicates precursors from Q1 scan that produce the characteristic fragment
• Low resolution (Q1 scan)
![Page 101: QStar Presentation 050503 · 2009. 11. 29. · Sample Preparation and Acquisiton. Orifice Plate Contamination Contaminant Buildup Around ... Use DHB Matrix Only QSTAR Target Bruker](https://reader036.fdocuments.us/reader036/viewer/2022081619/60fbeefe714ce04a877142e4/html5/thumbnails/101.jpg)
Precursor Ion Scanning
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Precursor Ion Scan
●●
Compound Tab MS Tab
Stores TOF m/z Range: Can Generate New
Precursor Scan From Other Fragment Ions
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Precursor Ion Scan
Resolution Tab Advanced Tab
Peak Hopping = Integer m/z ValuesProfile = Fractional m/z Values
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Precursor Ion Scan
500Da (1Da Step Size) x 0.02s Accumulation Time = 10 s Scan Time
●●
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300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700m/z, amu
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
8588
411.351
342.625
342.791
407.750
305.641415.749 514.192314.644 345.356
335.121 356.890442.821 519.940 693.587395.154
325.174418.902362.896
372.173
Beta Casein Trypsin DigestNegative Mode TOFMS
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Beta Casein Trypsin DigestNegative Mode Precursor of 79
300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700m/z, amu
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
7579
341.854
410.662
513.843
519.568423.310352.112
331.364
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Information-Dependent Acquision (IDA)
![Page 108: QStar Presentation 050503 · 2009. 11. 29. · Sample Preparation and Acquisiton. Orifice Plate Contamination Contaminant Buildup Around ... Use DHB Matrix Only QSTAR Target Bruker](https://reader036.fdocuments.us/reader036/viewer/2022081619/60fbeefe714ce04a877142e4/html5/thumbnails/108.jpg)
LCMS IDA Cycle1. TOFMS Survey Scan
2. Dependent Product Ion Scan-Peak Intensity-Charge State-m/z Range
3. New TOFMS Survey Scan-Dynamic Exclusion
4. Repeat Cycle Until End of LC Run
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Display With IDA Explorer
![Page 110: QStar Presentation 050503 · 2009. 11. 29. · Sample Preparation and Acquisiton. Orifice Plate Contamination Contaminant Buildup Around ... Use DHB Matrix Only QSTAR Target Bruker](https://reader036.fdocuments.us/reader036/viewer/2022081619/60fbeefe714ce04a877142e4/html5/thumbnails/110.jpg)
Rolling Collision Energy3
Col
lisio
n En
ergi
esR
ollin
g C
E
IDA CE Parameters.dll(Scripts Menu)
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Include and Exclude Lists
Can Import a Tab-Delimited List
Include List Ions Get First Priority in Selection for IDA
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Include and Exclude Lists
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Include and Exclude Lists
Exclude For Entire RunExclude For First 5 MinutesExclude From 14 to 16 Minutes
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Mass Difference (and Neutral-Loss) IDA
Induce Some In-Source Fragmentation For Neutral-Loss IDA
Can be Used to Detect
Isotope Pairs (ex. ICAT)
![Page 115: QStar Presentation 050503 · 2009. 11. 29. · Sample Preparation and Acquisiton. Orifice Plate Contamination Contaminant Buildup Around ... Use DHB Matrix Only QSTAR Target Bruker](https://reader036.fdocuments.us/reader036/viewer/2022081619/60fbeefe714ce04a877142e4/html5/thumbnails/115.jpg)
Troubleshooting
![Page 116: QStar Presentation 050503 · 2009. 11. 29. · Sample Preparation and Acquisiton. Orifice Plate Contamination Contaminant Buildup Around ... Use DHB Matrix Only QSTAR Target Bruker](https://reader036.fdocuments.us/reader036/viewer/2022081619/60fbeefe714ce04a877142e4/html5/thumbnails/116.jpg)
Troubleshooting Steps
1) Check nitrogen gas supply
2) Exit and restart Analyst QS
3) Stop Analyst Services and restart
4) Close Analyst QS and restart Windows
5) Deactivate and activate Hardware Profile
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Stop and Restart Analyst Services
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Deactivate and Reactivate the Hardware Profile
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Protein ID Software Tools
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Protein and PTM Identifications
• Individual Spectra– BioTools– Mascot
• IDA Data– BioTools– Mascot– Pro ID
![Page 121: QStar Presentation 050503 · 2009. 11. 29. · Sample Preparation and Acquisiton. Orifice Plate Contamination Contaminant Buildup Around ... Use DHB Matrix Only QSTAR Target Bruker](https://reader036.fdocuments.us/reader036/viewer/2022081619/60fbeefe714ce04a877142e4/html5/thumbnails/121.jpg)
Bioanalyst 1.1 Tutorial
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Pro ID Tutorial
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Mascot Tutorials
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Pro ID Database Searching
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Pro ID Script
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Data Dictionary
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Pro ID Search Results
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View Sequence
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Show Peptide ID Evidence
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View Selected Ions
![Page 131: QStar Presentation 050503 · 2009. 11. 29. · Sample Preparation and Acquisiton. Orifice Plate Contamination Contaminant Buildup Around ... Use DHB Matrix Only QSTAR Target Bruker](https://reader036.fdocuments.us/reader036/viewer/2022081619/60fbeefe714ce04a877142e4/html5/thumbnails/131.jpg)
Printing: Report Templates
![Page 132: QStar Presentation 050503 · 2009. 11. 29. · Sample Preparation and Acquisiton. Orifice Plate Contamination Contaminant Buildup Around ... Use DHB Matrix Only QSTAR Target Bruker](https://reader036.fdocuments.us/reader036/viewer/2022081619/60fbeefe714ce04a877142e4/html5/thumbnails/132.jpg)
Report Template Editor
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Report Template Margins
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Select Report Template For Printing
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Other Software Features
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Highlight Peak
Graph Information Window
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List Data
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List Data
Set Threshold For Peak Labeling
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Show File Information
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Show File Information
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Changing Graph Label Precision
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Trace Color
Main
Alternate(BPC)