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Operation Procedure for Sciex 4500/5500
Contact info: 901-448-7376(phone); dma6@uthsc.edu
General Policy
1. Please email dma6@uthsc.edu before you start using the LC/MS the first time. You must be
trained in order to use the LC/MS. No one is allowed to use other people’s account unless
authorized by SIF staff.
2. You may book time using Faces Scheduling website.
3. If you find any problem with instrument, DO NOT CONTINUE and please inform SIF staff asap.
I Operation Using Autosampler with Established Method
I-a Representative Biological Sample Preparation Procedure (reference only)
1. Place 50 µl of plasma or serum or other type of biological matrix in a plastic vial (0.5 mL).
2. Add 150 µl of cold (refrigerated) organic (ACN or MeOH) and vortex for 10 seconds.
3. Ice-cool the sample for 30 mins.
4. Centrifuge the sample to pellet out the precipitated proteins.
5. Take 150 µl of the supernatant and transfer to a HPLC vial or insert.
6. 10 µl on 4500 or 1 µL on 5500 is suggested inject volume to start with for LC/MS/MS analysis.
I-b LC-MS Preparation
1. When preparing mobile phase solution, degassing ahead of time is highly recommended.
2. Use a HPLC column in good working condition.
3. Make sure there are sufficient mobile phase solution and wash solution
4. If wash solution is low, please inform SIF staff
I-c Logging on the Workstation and Startup in Configure Mode
1. Enter your username and you password.
2. Click on “Analyst” icon on the desktop to start the program.
3. Open or create a user’s Project folder by clicking on Tools→Project→Create Project
4. Activate the instrument. Double click the Hardware Configuration and activate the LC-MS
profile. After a green check appears for selected profile, Close the window.
I-d Acquire Mode Operation
1. Enter Acquire mode and open Queue Manager window by clicking ‘View Queue’ icon
2. Click ‘Ready’ icon to make the LC-MS hardware ready
3. Click ‘Equilibrate’ icon and choose the LC-MS method to start the LC flow.
4. Place samples in the autosample, using 1mL vial, 1.5mL vial, or 96-well plate.
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5. Build Acquisition Batch using the Batch Editor window
a. Under Sample tab, choose LC-MS method from Method Editor
b. Click Add Set and then Add Samples. Build and save the Batch.
c. Under Submit tab, highlight the samples that need to be run, and click Submit
6. Check the Queue Manager window and click ‘Start Sample’ icon to start the LC-MS run
7. When finished, stop the Queue, and Standby the instrument.
8. Data can be processed under Explore mode or using MultiQuant software
II Manual Compound Optimization Using Syringe Pump Infusion (Method Development).
II-a Sample Requirements
1. For compound profiling, prepare your sample solution at or less than 1µg/ml in methanol or
acetonitrile. (NOT 1 mg/ml, WHICH IS TOO MUCH AND WILL CLOG THE ION PATH).
2. Sample purity plays an important role in speed and accuracy of analysis as well as method
ruggedness. Endogenous matrix component such as proteins, lipids, salts, and extracellular
materials should be removed via precipitation, extraction, filtration, centrifugation, or any
methods possible.
3. Compounds to be avoid
◊ Salts can interfere with ionization and can cluster to complicate spectrum (but also aid in
identification).
◊ Strong based or quaternary amines can interfere with positive mode analytes, e.g.
Triethylamine (TEA).
◊ Acids-sulfuric/sulfonic acids and TFA interfere in negative mode experiments
◊ Phosphate buffer and non-volatile ion pairing agents (e.g. SDS) can cause severe suppression
and complex spectra.
◊ Dimerization ([2M+H]+) can occur at high concentration, leading to non-linearity during
quantitation.
◊ Dimer signal at m/z=(MW*2)+1 can cause non-linearity at high concentrations.
II-b Logging on the Workstation and Startup
1. Enter your username and you password.
2. Click on “Analyst” icon on the desktop to start the program.
3. Create a user’s Project folder by clicking on Tools→Project→Create Project (enter a project
name).
4. Activate the instrument. Double click the Hardware Configuration and activate the Mass Spec
Only profile. After a green check appears for selected profile, Close the window.
II-c Introducing Your Sample (direct infusion)
1. Fill the 1.0mL Hamilton syringe with methanol to clean infusion tubing.
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2. Fill the syringe with your sample (1ug/mL or lower); place the syringe on the built-in syringe
pump; turn on the syringe pump from Tuning mode window or press the button next to the
syringe pump. The default syringe flow rate is 7 or 10 µl/min. Start the Syringe pump.
II-d Run Q1 Scan
Q1 Scan is an MS full scan (start-stop) where the first quadrupole Q1 acts as single MS analyzer and
the third quadrupole Q3 transmits all ion toward the detector region. Q1 scan is used primarily for
identification of precursor/parent ion.
1. In the navigator bar, click on Tune. If open, close the Tune Method Editor Window.
2. Double click on Manual Tuning to re-open the Tune Method Editor Window. You should hear
the gases turned on (depending on previous status, the gas maybe on already) as the instrument
becomes active for use.
3. In Scan Type, select Q1 Scan
4. Enter a Start (amu) mass value, Stop (amu) value.
5. Select suitable Polarity. (Positive or Negative)
6. Set Duration Time (min) to 5.
7. In the Source Gas tab, set NEB to 8, CUR to 20.
8. Other parameters use default settings for now.
9. Click Start to monitor the MS spectra.
10. Click Acquire to store the MS spectra data in a file. You can open and check the data later.
11. Click Stop to finish before duration time is reached.
II-e Run Product Ion Scan
Product Ion Scan is an MS/MS scan where the first quadrupole Q1 is fixed and the third quadrupole
Q3 sweeps a range. It is an experiment that will search for all of the products of a particular
precursor/parent ion.
1. In the navigator bar, click on Tune. If open, close the Tune Method Editor Window
2. Double click on Manual Tuning to re-open the Tune Method Editor Window. You should hear
the gases turned on (depending on previous status, the gas maybe on already) as the instrument
becomes active for use.
3. In Scan Type, select Product Ion Scan.
4. In the Products of filed, enter one of the precursor ions observed in the Q1 scan above.
5. Enter a Start (amu) mass value of 40, Stop (amu) value of 10 mass units above the selected
precursor/parent ion and Time (sec) of one scan cycle (such as:3).
6. Select suitable Polarity. (Positive or Negative)
7. Set Duration Time (min) to 5.
8. In the Source Gas tab, set NEB to 8, CUR to 20, set CAD gas to 4.
9. In the Compound tab, set the DP to 65, set CE as 10, use default for other settings now.
10. Click Start to monitor the MS/MS spectra.
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11. After scaning has started, Increase the CE 5v at a time until 80v and observe how fragmentation
patter shifts from high mass fragments to low mass fragments. The spectrum displayed will
correlate to the CE entered. Choose a suitable CE for your compound. Take note of suitable
product ions to be used for MRM transition.
12. Click Acquire to store the MS/MS spectra data in a file. You can open the file in the future.
13. Click Stop to finish before duration time is reached.
II-f Compound Optimization
MRM is an MS/MS technique where the first quadrupole Q1 is fixed and the third quadrupole Q3 is
also fixed. MRM is used for quantitation. Analyst software provides Compound Optimization to
optimize DP, CE, CXP parameters quickly for each MRM transition.
1. In the navigator bar, click on Tune. If open, close the Tune Method Editor Window
2. Double click on Manual Tuning to re-open the Tune Method Editor Window. You should hear
the gases turned on (depending on previous status, the gas maybe on already) as the instrument
becomes active for use.
3. Click Compound Optimization. Choose Infusion as Inlet; and MS/MS Analysis;
MW Ion search window ± 0.5 Da; Resolution Unit;
Product Ion choose User Specified; Resolution Unit
Select Polarity: Positive or Negative suitable for the compound
Input Compound Name, Q1 Mass, and Q3 Mass. Start automatic optimization
4. After it is done, it will indicate either optimization is successful or not. If successful, note the
optimized DP, CE, CXP values and use the settings to create LC-MS method.
5. Stop the syringe pump.
II-g Explore MS Data
1. Select your project folder.
2. Click on Explore on the navigator bar. Open the data file to display your MS spectrum.
II-h Finishing Up
1. Stop the Queue. Put instrument at Standby
2. De-activate the instrument. Double click the Hardware Configuration and de-activate the Mass
Spec Only profile. After a green check disappears for selected profile, Close the window.
Leaving the instrument
1. Exit the Analyst program.
2. Log out of the computer (DO NOT shut down the computer)
3. Clean up the instrument table, put the syringe in its box, and switch off the syringe pump.
4. Please inform any problem to SIF staff
