OHS026 Safe Work Procedure

OHS026
Safe Work Procedure
Faculty/Division
DVC Research
Document number
SWP02.13
School/ Divisional Unit
BMSF / Analytical Centre
Initial Issue date
2/9/08
Current version
1.0.0.1
Current Version
Issue date
Next review date
The Writing Safe Work Procedures Guideline (OHS027) should be consulted to assist in the completion of this form.
Safe Work Procedure Title and basic description
Title: Calibration and sample analysis by nanoLC-MS/MS on the LTQ-FT Ultra
Description: Associated risk assessment title and location:RA02.16
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Safe Work Procedure
Date Effective: 01/01/2007
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Current Version: 1.2, 15/08/2007
Describe the activity or process
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Safe Work Procedure
Date Effective: 01/01/2007
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Solvent/sample preparation
•
Samples and mobile phase mixtures should be prepared in the BMSF wet lab areas (M304/M310) or in the operator’s
laboratory prior to the commencement of this procedure.
Calibration of the LTQ-FT Ultra
1.
Check the instrument is not running a sample (Xcalibur). If the instrument is running a sample, stop/pause queue.
Note: Stopping/pausing the queue while a sample is running will not stop its run. The queue will pause after the sample has
finished.
2.
Stop the LC flow in Chromeleon. In control menu, unclick ‘Monitor Only’ and stop flow by clicking on the Stop button ().
Loading pump and micropump should slowly go ‘Off’. Under the Column Comp tab, click on ‘Splitter Details’ and change
‘Flow Control Mode’ to ‘Off’ and ‘Control Mode’ to ‘Off’ (if it isn’t already off).
3.
Wind the LC column back from the ion source and pull the electrospray stand out.
4.
Unplug the voltage (red line) and unscrew the gas line (clear tubing).
Warning: Ensure the voltage line is unplugged before proceeding to the next step.
5.
Carefully unscrew the LC column assembly and place to the side.
Caution: The LC column tip is sharp so avoid touching the tip to prevent injury. Wear safety glasses. Also avoid damaging the
column but ensuring that the tip does not hit any other surfaces and that it does not slide back into the column sleeve/holder
while placing it aside.
6.
Screw on the electrospray needle block (sits on the right) and plug in the voltage line (red line).
7.
Take a Proxeon nanospray tip and cut the end to ~2cm using a ceramic tile (score and break). Pipette 2µL of the calibration
solution (contains cholesterol 195, MRFA peptide (singly charged at 524 and doubly charged at 262) and ultramark 1650) and
flick the tip to get the solution to the point of the tip.
Caution: The Proxeon nanospary tips are very sharp so handle with care (particularly when flicking) and wear safety glasses.
Dispose any cut parts of the Proxeon nanospray tips appropriately- refer to Clean up and Waste disposal section below.
8.
Insert the Proxeon nanospray tip into the nanospray block until ~1cm protrusion and screw (not too tight) into place. Push the
electrospray stage towards the ion source. Do this carefully as we do not want to hit the ion source particularly if the needle is
protruding too far.
9.
Adjust the tip position up/down/in/out/left/right using the knobs at the back of the stage and position the tip to the side of the
ion source. Use cameras as a guide. Quickly hit the tip on the side of the ion source to break it and create an opening for
liquid flow.
Caution: The ion source is very hot. Do not leave the needle on the source for too long when breaking the tip otherwise the
needle will melt.
10. To ensure liquid flow of the Proxeon nanospray tip, apply some pressure using the syringe pump attached to the nanospray
block. Once liquid is flowing, position the tip in front of the source.
11. To start the flow, open the LTQ tune software (located on desktop) and open the ‘Nano_LC_for calibration’ file. Press
‘Operate’ (play/pause button).
12. Select the NSI source (from MS diagram) and change the ion spray voltage to 0.8V and the tube lens to 175 for calibration (for
nanospray MS/MS analysis, the tube lens should be changed back to 125).
13. To select/change the scan mode, press the define scan button (button with the dots/circles) and select FT (for calibration of
FT). Change the scan range to 190-1900m/z. The default resolution should be 10000. Press ‘Apply’.
Note: The scan range should always be within a factor of 10; any bigger will make the FT scanning not optimal.
14. Go to the Control menu and select ‘Calibration’.
15. Select the FT manual tab
Note: Do not use ‘Automatic’ because the instrument will calibrate using a long list of parameters.
16. Under ‘Mass List’, select ‘Calmix +ve (factory)’. Under ‘Check’, select ‘Mass’ and press ‘Start’.
Note: This will measure the mass of ions and work out an average error. If this fails then this step may be repeated or the tube
lens could be changed to 190 or 150. If this still does not work, go to ‘Define Scan’ and select Ion Trap and adjust the tip
position. It is important that the correct amounts of ions are entering the FT. If this step continues to fail, try calibrating (next
step).
17. When the calibration mass check is OK (having an error of less than 2ppm is good), go to the ‘Calibrate’ section and select
‘Mass’ and press ‘Start’. When the calibration is complete, the readout will say ‘Calibration Finished’. Record the calibration
in a Word document and save.
18. Following calibration, under ‘Check’, select ‘Ion Transmission’ and press ‘Start’.
Note: Ion transmission checks that the transfer of ions from the ion trap to the FT cell is OK. For this to occur successfully,
check that there is a good signal before calibration. If successful, Calibration should be OK.
LC-MS/MS
1. After calibration, swap the nanaspray block for the LC column assembly. Do this by winding the nanospray block back and
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pull the electrospray stand away from the ion source.
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Safe Work
Procedure
Date Effective: 01/01/2007
2. Unplug
the voltage line (red line).
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Current Version: 1.2, 15/08/2007
3. Unscrew the tip holder and pull out the Proxeon nanospray tip and dispose of appropriately. Unscrew and remove the
List all resources required including plant, chemicals,
personal protective clothing and equipment, etc
Manufacturers detailed manuals and safety instructions are available online (//files1med/bmsf/BMSF_all/MANUALS/Mass Specs) or printed copies are located on shelves near equipment.
[For see LTQ-FT Ultra.pdf and related manuals].
1.
Equipment
•
NanoES spray capillaries/tips (Medium) (Proxeon Biosystems, Cat. No. ES380)
•
2.5µL Eppendorf pipette
•
GELoader Tips, 0.5-20µL (Eppenforf, Cat. No. 0030 001.222)
•
LC column
2.
Substances/samples
•
LTQ-FT calibration solution
•
Protein samples for LC-MS/MS
•
Buffer A: 2% acetonitrile/0.1 % formic acid/98% deionised water
•
Buffer B: 80% acetonitrile/0.1% formic acid/20% deionised water
•
Buffer D: 0.05% Heptafluorobutyric acid/2% acetonitrile/98% deionised water
3.
PPCE
•
•
•
Safety Glasses
Lab Coat
Gloves (No gloves permitted on keyboard or instrument)
List potential hazards and risk controls including specific
precautions required
Key Hazards
Splash/spill when filling solvent reservoirs
Moving parts
Risks from Hazards
Physical injury- Eye and body
Physical injury- Hand
Sharps- Cuts by handling the Proxeon
capillaries and the LC column
Physical injury- Eye and body parts
Hot surfaces
Electrical
High Magnetic Field
Burns
Shock
Death
Control Measure including PPE
Safety glasses, lab coat and gloves
Training – make sure it is not in operate
mode, remove sample tray to load
samples and do not access samples while
instrument is injecting a sample.
Safety Glasses, gloves and lab coat.
Training for safe handling. Proper disposal
of capillaries into yellow Sharps bins
immediately after use.
Don’t touch hot surface
Don’t touch ESI probe
Keep >2m away from Magnet
List emergency shutdown instructions
***Follow recommended procedure near instrument or turn mains power off in REAL EMERGENCY***. (Magnetic field CANNOT be
discharged without special equipment from manufacturer).
1.
Inform supervisor, lab manager, instrument manager and first aid officer: Mark Raftery (M309; x51892) and Lydia Morris
(M302; x58702)
2.
For large scale emergencies dial x56666
3.
For electrical or fire hazards, fire extinguishers are located outside M307 and in M304
4.
For any injuries requiring first aid (e.g. sharps), the nearest first aid kit is located in M310 or M304
5.
Eye wash stations are located in M304 & M312
6.
Safety shower located in M304
7.
Instrument should be left on in standby mode when not being used
List clean up and waste disposal requirements
1.
Dispose of all the Proxeon nanospray tips and their cut fragments in sharps bins provided.
2.
Following LC-MS/MS, if samples do not need to be stored for further use, they should be discarded in biological waste bins.
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Safe Work Procedure
Date Effective: 01/01/2007
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List legislation, standards and codes of practice used in
the development of the SWP
-
Supervisory approval, training, and review
Supervisor: Mark Raftery or Valerie Wasinger
Signature:
Plant custodian:
Signature
List competency required – qualifications, certificates, licencing, training - eg course or instruction:
•
Induction to BMSF facility by Lydia Morris.
•
LTQ-FT training required by one of the above staff members and supervision required for the task until deemed
competent by an authorised staff member (or delegate).
SWP review date:
Responsibility for SWP review:
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Safe Work Procedure
Date Effective: 01/01/2007
Uncontrolled document when printed
Current Version: 1.2, 15/08/2007