QSTAR Operation May 3, 2005 Bob Seward
Presentation CD Contents
QSTAR Hardware
QSTAR Schematic 10-7 torr
QSTAR Schematic Chernushevich et al, JMS 2001, 36: 849-865
Curtain Plate ESI Source
ESI Source Curtain Gas Curtain Plate
QSTAR Mass Ranges Upper m/z Ranges QSTAR 1 QSTAR 2 TOFMS 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
Ring Skimmer Removed
Skimmer
Q0 Source Removed: Q0
Quadrupole Rail Q0 Q1 Q2 - Enclosed To Maintain CAD Gas Pressure
Decreasing Pressure Stubbies and Q1
Q2 and CAD Gas Supply
Q2 Exit Lens
DC Quad Lens: Directs Ion Beam Into TOF
Nanospray
Nanospray Source Cameras Side Center XYZ Stage
Nanospray Tip Positioning 1: Be Sure That QSTAR Is In Standby Mode Move Tip Close To Orifice
Nanospray Tip Positioning 2 Move Nanospray Tip Off Axis Center Side Off Axis
Nanospray Tip Positioning 3 Move Tip Back Center Side Good Spraying Distance
Sample Preparation and Acquisiton
Orifice Plate Contamination Contaminant Buildup 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 [Glu 1 ]-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% NH 4 OH (v/v, using 30% NH 4 OH 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 Hardware Setup, Printing Configuration, Security Manual Acquisition Information Dependent Acquisition (IDA) Data Analysis
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
New Project Icon Creating a Project
Creating a Project Type in Project Name Press OK: D:\PE Sciex Data\Projects\My Project Data Folder Acquisition Methods Folder
Startup: Open Project and Tune Mode (2) Select Tune Mode (1) Open Project Instrument Status Indicator: Green -Ready Yellow -Standby Red -Fault
Make Sure That The Instrument Is In Standby Mode Before Touching the Source!
Startup: Open the Queue Manager (3) View Queue Standby / Ready Mode Queue Manager Can Be Minimized
Startup: Open Manual Tuning Window Manual Tune Icon 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
* User Adjustable Instrument Settings * * * * * 10-7 torr
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 (3) Open File (2) Manual Tune Window Open (1) Ready Mode
Product Ion Mode
Set Acquisiton Parameters: Product Ion Mode Compound Tab MS Tab
Set Acquisiton Parameters: Resolution & Advanced MS Tabs Low Q1 Resolution: - Increased sensitivity - Transmit isotope envelope - TOF resolution unaffected Resolution Tab Advanced MS Tab
Ion Transmission Efficiency
Quadrupole Transmission Windows, TOFMS (RF-Only) Min Max m/z 400-1700 Max m/z Min m/z > ~5 350-1700 (2 Hops ) Quadrupole Hops To Cover m/z Range 80-1700 (3 Hops )
QSTAR Schematic 10-7 torr
Orthogonal Injection TOF slice of ions injected Detector Ion Beam from Quad
Duty Cycle of Orthogonal Injection TOF Pulse 10 ev X = width of ejected beam slice TOF Cycle L = distance traveled by ions between pulses Duty cycle = x/l = 4% (m/z 100) = 12% (m/z 1000) = 26% (m/z 5000)
TOF Pulser Frequency Changes With Maximum m/z Slower Frequency = Lower Transmission Efficiency Min Max m/z 400-2000 400-2100 TOF Analyzer Waits Longer Between Cycles Ion Beam From Quad is Lost During This Time Pulser Frequency Range: 3-20 KHz
Q2 Pulsing (Enhancing)
Enhancing Enhance a m/z Range Enhance a Single m/z
QSTAR Pulsar: Pulsing ON Q2 collision cell Ions are Stored in Q2, then Pulsed into the TOF
Q2 Exit Lens
Enhancing a Single m/z IRD & 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-Click Next to % Column
MALDI Analysis
MALDI Target and Adapter Use DHB Matrix Only QSTAR Target Bruker Adapter
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 Server Start 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 Peak Hopping = Integer m/z Values Profile = Fractional m/z Values Resolution Tab Advanced Tab
Precursor Ion Scan 500Da (1Da Step Size) x 0.02s Accumulation Time = 10 s Scan Time
Beta Casein Trypsin Digest Negative Mode TOFMS 88 85 342.625 411.351 80 75 70 65 60 55 50 342.791 45 40 35 30 25 20 15 10 305.641 314.644 345.356 335.121 356.890 325.174 362.896 407.750 415.749 514.192 395.154 442.821 519.940 418.902 372.173 693.587 5 0 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700 m/z, amu
Beta Casein Trypsin Digest Negative Mode Precursor of 79 79 75 341.854 70 65 60 55 410.662 50 45 40 35 30 25 20 513.843 15 10 331.364 352.112 423.310 519.568 5 0 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700 m/z, amu
Information-Dependent Acquision (IDA)
LCMS IDA Cycle 1. 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 Energy IDA CE Parameters.dll (Scripts Menu) Rolling CE 3 Collision Energies
Include and Exclude Lists Include List Ions Get First Priority in Selection for IDA Can Import a Tab-Delimited List
Include and Exclude Lists
Include and Exclude Lists Exclude For Entire Run Exclude For First 5 Minutes Exclude 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
Graph Information Window Highlight Peak
List Data
List Data Set Threshold For Peak Labeling
Show File Information
Show File Information
Changing Graph Label Precision
Trace Color Main Alternate (BPC)