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)