QSTAR Operation
May 3, 2005
Bob Seward
Presentation CD Contents
QSTAR Hardware
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QSTAR Schematic
QSTAR Schematic
Chernushevich et al, JMS 2001, 36: 849-865
ESI Source
Curtain Plate
ESI Source
Curtain Gas
Curtain Plate
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
With Turbo IonSpray Source
TOF Analyzer
Turbo Pumps
ESI Source With Curtain Plate
Curtain Plate Removed
Source Removed Skimmer Facing Up
Skimmer Removed
Ring
Skimmer
Q0
Source Removed: Q0
Q0 Q1 Q2 - EnclosedTo Maintain CAD
Gas Pressure
Quadrupole Rail
Stubbies and Q1
Decreasing Pressure
Q2 and CAD Gas Supply
Q2 Exit Lens
DC Quad Lens: Directs Ion Beam Into TOF
Nanospray
Nanospray Source Cameras
CenterSide
XYZ Stage
Move TipClose ToOrifice
Nanospray Tip Positioning 1: Be Sure That QSTAR Is In Standby Mode
Center Side
Off Axis
Nanospray Tip Positioning 2Move Nanospray Tip Off Axis
Nanospray Tip Positioning 3Move Tip Back
Center Side
Good Spraying Distance
Sample Preparation and Acquisiton
Orifice Plate Contamination
ContaminantBuildup Around
The Orifice
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)
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
Wash The Capillary With Solvent (50%ACN)
Dry The Capillaries Overnight Before Pulling The Nanospray Tips
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
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)
Analyst QS Software
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
Analyst QS Overview
Manual Acquisition
Information Dependent Acquisition (IDA)
Data Analysis
Hardware Setup, Printing Configuration, Security
Analyst QS Overview
Navigation Bar:Most functions accessible from toolbars and menus.Can be closed to save screen space.
Analyst QS Overview
Configure Mode: Security Configuration
Analyst QS Overview
Help Topics
Creating a Project
“New Project” Icon
Creating a Project
Type in Project Name – Press OK:D:\PE Sciex Data\Projects\My Project
Data FolderAcquisition Methods Folder
(1) Open Project(2) Select Tune Mode
Startup: Open Project and Tune Mode
Instrument Status Indicator:Green -ReadyYellow -StandbyRed -Fault
Make Sure That The Instrument Is In Standby Mode Before
Touching the Source!
(3) View Queue Standby / Ready Mode
Startup: Open the Queue Manager
Queue Manager Can Be Minimized
“Manual Tune” Icon
Startup: Open Manual Tuning Window
Ready Mode
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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Instrument Settings
***
**
*
*
* User Adjustable
TOF MS Acquisition
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
Set to Nanospray and Adjust “TOF Offset” to Correct Value
There Are Separate “TOF Offset” Values
For Negative And Positive Modes
Set Acquisition Parameters: Compound & Advanced MS Tabs
Compound Tab Advanced MS Tab
Save The Acquisition Method
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
Open Acquisition Method
(1) Ready Mode(2) Manual Tune Window Open
(3) Open File
Product Ion Mode
Set Acquisiton Parameters: Product Ion Mode
Compound Tab MS Tab
Set Acquisiton Parameters: Resolution & Advanced MS Tabs
Advanced MS TabResolution Tab
Low Q1 Resolution:- Increased sensitivity- Transmit isotope envelope- TOF resolution unaffected
Ion Transmission Efficiency
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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QSTAR Schematic
Ion Beamfrom Quad
Detector
“slice” of ions injected
Orthogonal Injection TOF
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
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
Q2 Pulsing (Enhancing)
Enhancing
Enhance a Single m/z
Enhance a m/z Range
Ions are Stored in Q2, then Pulsed into the TOF
Q2 collision cell
QSTAR Pulsar: Pulsing ON
Q2 Exit Lens
Enhancing a Single m/zIRD & IRW are Optimized for that m/z
No effect on the Quadrupole Transmission Windows
Enhance All
More Quadrupole Transmission Windows are Generated
IRD & IRW are Optimized for Each Window
No Enhancing (70-1800)
Enhance All (70-1800)
Enhance All
Enhancing: Glu-Fibrinopeptide B
No Enhance Enhance All Enhance 785.8
Increasing Quadrupole Residence Time at Higher Mass
Changing CE For Individual Quadrupole Transmission Windows
Right-ClickNext to %Column
MALDI Analysis
MALDI Target and AdapterUse DHB Matrix Only
Bruker AdapterQSTAR Target
MALDI Source
Laser Guide
Installing the MALDI Target
Target
Installing the MALDI Target
oMALDI Server Software
• Controls laser and sample target
• Sample acquisition controlled by Analyst QS
• Start acquisition first – then switch laser on
oMALDI ServerStart From Desktop Icon
Pumping Down The MALDI Source
Turns Bright Green When Vacuum is
Ready
Removing The MALDI Plate
Laser Control
Pulse Rate: 20
Attenuator: 20,000
Laser On/Off
Positioning The Target and Sample
Positioning The Sample
TOF Calibration
Select Two Peaks – Right Click – Re-Calibrate TOF
Select Calibration File
Select Calibration File
Calculate New Calibrations
Calculate New Calibrations
Calibrate Spectrum
Calibrate Spectrum
Apply to Entire File
For Highest Accuracy: Calibrate Instrument and Acquire Samples with the Same TOF Pulser Frequency
Recalibration of a Spectrum
Apply Calibrations to Sample
Uncheck Boxes
Save As Recalibrated File
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)
Precursor Ion Scanning
Precursor Ion Scan
●●
Compound Tab MS Tab
Stores TOF m/z Range: Can Generate New
Precursor Scan From Other Fragment Ions
Precursor Ion Scan
Resolution Tab Advanced Tab
Peak Hopping = Integer m/z ValuesProfile = Fractional m/z Values
Precursor Ion Scan
500Da (1Da Step Size) x 0.02s Accumulation Time = 10 s Scan Time
●●
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
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
Information-Dependent Acquision (IDA)
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
Display With IDA Explorer
Rolling Collision Energy3
Col
lisio
n En
ergi
esR
ollin
g C
E
IDA CE Parameters.dll(Scripts Menu)
Include and Exclude Lists
Can Import a Tab-Delimited List
Include List Ions Get First Priority in Selection for IDA
Include and Exclude Lists
Include and Exclude Lists
Exclude For Entire RunExclude For First 5 MinutesExclude From 14 to 16 Minutes
Mass Difference (and Neutral-Loss) IDA
Induce Some In-Source Fragmentation For Neutral-Loss IDA
Can be Used to Detect
Isotope Pairs (ex. ICAT)
Troubleshooting
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
Stop and Restart Analyst Services
Deactivate and Reactivate the Hardware Profile
Protein ID Software Tools
Protein and PTM Identifications
• Individual Spectra– BioTools– Mascot
• IDA Data– BioTools– Mascot– Pro ID
Bioanalyst 1.1 Tutorial
Pro ID Tutorial
Mascot Tutorials
Pro ID Database Searching
Pro ID Script
Data Dictionary
Pro ID Search Results
View Sequence
Show Peptide ID Evidence
View Selected Ions
Printing: Report Templates
Report Template Editor
Report Template Margins
Select Report Template For Printing
Other Software Features
Highlight Peak
Graph Information Window
List Data
List Data
Set Threshold For Peak Labeling
Show File Information
Show File Information
Changing Graph Label Precision
Trace Color
Main
Alternate(BPC)