QSTAR Operation. May 3, Bob Seward

Transcription

1 QSTAR Operation May 3, 2005 Bob Seward

2 Presentation CD Contents

3 QSTAR Hardware

4 QSTAR Schematic 10-7 torr

5 QSTAR Schematic Chernushevich et al, JMS 2001, 36:

6 Curtain Plate ESI Source

7 ESI Source Curtain Gas Curtain Plate

8 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

9 With Turbo IonSpray Source

10 TOF Analyzer

11 Turbo Pumps

12 ESI Source With Curtain Plate

13 Curtain Plate Removed

14 Source Removed Skimmer Facing Up

15 Ring Skimmer Removed

16 Skimmer

17 Q0 Source Removed: Q0

18 Quadrupole Rail Q0 Q1 Q2 - Enclosed To Maintain CAD Gas Pressure

19 Decreasing Pressure Stubbies and Q1

20 Q2 and CAD Gas Supply

21 Q2 Exit Lens

22 DC Quad Lens: Directs Ion Beam Into TOF

23 Nanospray

24 Nanospray Source Cameras Side Center XYZ Stage

25 Nanospray Tip Positioning 1: Be Sure That QSTAR Is In Standby Mode Move Tip Close To Orifice

26 Nanospray Tip Positioning 2 Move Nanospray Tip Off Axis Center Side Off Axis

27 Nanospray Tip Positioning 3 Move Tip Back Center Side Good Spraying Distance

28 Sample Preparation and Acquisiton

29 Orifice Plate Contamination Contaminant Buildup Around The Orifice

30 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)

31 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

32 Wash The Capillary With Solvent (50%ACN)

33 Dry The Capillaries Overnight Before Pulling The Nanospray Tips

34 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 (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

35 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)

36 Analyst QS Software

37 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

38 Analyst QS Overview Hardware Setup, Printing Configuration, Security Manual Acquisition Information Dependent Acquisition (IDA) Data Analysis

39 Analyst QS Overview Navigation Bar: Most functions accessible from toolbars and menus. Can be closed to save screen space.

40 Analyst QS Overview Configure Mode: Security Configuration

41 Analyst QS Overview Help Topics

42 New Project Icon Creating a Project

43 Creating a Project Type in Project Name Press OK: D:\PE Sciex Data\Projects\My Project Data Folder Acquisition Methods Folder

44 Startup: Open Project and Tune Mode (2) Select Tune Mode (1) Open Project Instrument Status Indicator: Green -Ready Yellow -Standby Red -Fault

45 Make Sure That The Instrument Is In Standby Mode Before Touching the Source!

46 Startup: Open the Queue Manager (3) View Queue Standby / Ready Mode Queue Manager Can Be Minimized

47 Startup: Open Manual Tuning Window Manual Tune Icon Ready Mode

48 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

49 * User Adjustable Instrument Settings * * * * * 10-7 torr

50 TOF MS Acquisition

51 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

52 Set to Nanospray and Adjust TOF Offset to Correct Value There Are Separate TOF Offset Values For Negative And Positive Modes

53 Set Acquisition Parameters: Compound & Advanced MS Tabs Compound Tab Advanced MS Tab

54 Save The Acquisition Method

55 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

56 Open Acquisition Method (3) Open File (2) Manual Tune Window Open (1) Ready Mode

57 Product Ion Mode

58 Set Acquisiton Parameters: Product Ion Mode Compound Tab MS Tab

59 Set Acquisiton Parameters: Resolution & Advanced MS Tabs Low Q1 Resolution: - Increased sensitivity - Transmit isotope envelope - TOF resolution unaffected Resolution Tab Advanced MS Tab

60 Ion Transmission Efficiency

61 Quadrupole Transmission Windows, TOFMS (RF-Only) Min Max m/z Max m/z Min m/z > ~ (2 Hops ) Quadrupole Hops To Cover m/z Range (3 Hops )

62 QSTAR Schematic 10-7 torr

63 Orthogonal Injection TOF slice of ions injected Detector Ion Beam from Quad

64 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)

65 TOF Pulser Frequency Changes With Maximum m/z Slower Frequency = Lower Transmission Efficiency Min Max m/z TOF Analyzer Waits Longer Between Cycles Ion Beam From Quad is Lost During This Time Pulser Frequency Range: 3-20 KHz

66 Q2 Pulsing (Enhancing)

67 Enhancing Enhance a m/z Range Enhance a Single m/z

68 QSTAR Pulsar: Pulsing ON Q2 collision cell Ions are Stored in Q2, then Pulsed into the TOF

69 Q2 Exit Lens

70 Enhancing a Single m/z IRD & IRW are Optimized for that m/z No effect on the Quadrupole Transmission Windows

71 Enhance All More Quadrupole Transmission Windows are Generated IRD & IRW are Optimized for Each Window No Enhancing ( ) Enhance All ( )

72 Enhance All

73 Enhancing: Glu-Fibrinopeptide B No Enhance Enhance All Enhance 785.8

74 Increasing Quadrupole Residence Time at Higher Mass

75 Changing CE For Individual Quadrupole Transmission Windows Right-Click Next to % Column

76 MALDI Analysis

77 MALDI Target and Adapter Use DHB Matrix Only QSTAR Target Bruker Adapter

78 MALDI Source Laser Guide

79 Installing the MALDI Target Target

80 Installing the MALDI Target

81 omaldi Server Software Controls laser and sample target Sample acquisition controlled by Analyst QS Start acquisition first then switch laser on

82 omaldi Server Start From Desktop Icon

83 Pumping Down The MALDI Source Turns Bright Green When Vacuum is Ready

84 Removing The MALDI Plate

85 Laser Control Pulse Rate: 20 Attenuator: 20,000 Laser On/Off

86 Positioning The Target and Sample

87 Positioning The Sample

88 TOF Calibration

89 Select Two Peaks Right Click Re-Calibrate TOF

90 Select Calibration File

91 Select Calibration File

92 Calculate New Calibrations

93 Calculate New Calibrations

94 Calibrate Spectrum

95 Calibrate Spectrum

96 Apply to Entire File For Highest Accuracy: Calibrate Instrument and Acquire Samples with the Same TOF Pulser Frequency

97 Recalibration of a Spectrum

98 Apply Calibrations to Sample Uncheck Boxes

99 Save As Recalibrated File

100 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)

101 Precursor Ion Scanning

102 Precursor Ion Scan Compound Tab MS Tab Stores TOF m/z Range: Can Generate New Precursor Scan From Other Fragment Ions

103 Precursor Ion Scan Peak Hopping = Integer m/z Values Profile = Fractional m/z Values Resolution Tab Advanced Tab

104 Precursor Ion Scan 500Da (1Da Step Size) x 0.02s Accumulation Time = 10 s Scan Time

105 Beta Casein Trypsin Digest Negative Mode TOFMS m/z, amu

106 Beta Casein Trypsin Digest Negative Mode Precursor of m/z, amu

107 Information-Dependent Acquision (IDA)

108 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

109 Display With IDA Explorer

110 Rolling Collision Energy IDA CE Parameters.dll (Scripts Menu) Rolling CE 3 Collision Energies

111 Include and Exclude Lists Include List Ions Get First Priority in Selection for IDA Can Import a Tab-Delimited List

112 Include and Exclude Lists

113 Include and Exclude Lists Exclude For Entire Run Exclude For First 5 Minutes Exclude From 14 to 16 Minutes

114 Mass Difference (and Neutral-Loss) IDA Induce Some In-Source Fragmentation For Neutral-Loss IDA Can be Used to Detect Isotope Pairs (ex. ICAT)

115 Troubleshooting

116 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

117 Stop and Restart Analyst Services

118 Deactivate and Reactivate the Hardware Profile

119 Protein ID Software Tools

120 Protein and PTM Identifications Individual Spectra BioTools Mascot IDA Data BioTools Mascot Pro ID

121 Bioanalyst 1.1 Tutorial

122 Pro ID Tutorial

123 Mascot Tutorials

124 Pro ID Database Searching

125 Pro ID Script

126 Data Dictionary

127 Pro ID Search Results

128 View Sequence

129 Show Peptide ID Evidence

130 View Selected Ions

131 Printing: Report Templates

132 Report Template Editor

133

134

135

136 Report Template Margins

137

138 Select Report Template For Printing

139 Other Software Features

140 Graph Information Window Highlight Peak

141 List Data

142 List Data Set Threshold For Peak Labeling

143 Show File Information

144 Show File Information

145 Changing Graph Label Precision

146 Trace Color Main Alternate (BPC)