LCMS-325 TQ Operation

1. LCMS-325 TQ Operation Chem 115 Paul Hrvatin Winter 2013

2. LCMS-325 TQ Operation • Make sure the solvent reservoirs are sufficiently filled with HPLC grade solvent. • Open System Control if it is not already open by clicking on the top and most left icon (Figure 1) on the tool bar at the top of the monitor. This opens up the Quad View (Figure 2). Make sure you are using view III (Figure 3) so that the proper quadrants are viewable. Make sure the correct method is loaded. This software should look familiar because it is similar to that used for the HPLC experiment. The desired method for running the ubiquitin samples is ‘MS-115’. It is a 22 minute method that allows for all 9 injections of the LCMS experiment with one extra 2 minute time block extension. However, the ‘Chem115 MeOH’ method, which is only a 4 minute method that is used in the shutdown protocol; is shown loaded in Figure 4. Changing methods is done by activating the desired method under the file tab of System Control. • Click Turn Spray On in the Quad view (Figure 5) to turn on the gases and heaters. Once the temperature reaches 75% of the set value, activate the pumps by navigating to that window using the Windows tab under System Control. Figure 1 Figure 2 Figure 3 Figure 4

3. LCMS-325 TQ Operation 3 cont. The spray icon now has a red stream coming from it (Figure 6) • A few minutes is required for the temperature to stabilize, load your sample into the sample loop during this time making sure the valve is in the load position. • Under the file tab In System Control, open up the sample list and set the data file and destination. Toggle back to the Quad View using the windows tab. • Once the API and 325 indicate they are ready (Figure 7), its time turn on the analyzer. The button turns green when it has been activated (Figure 8). • Toggle back to the sample list using the windows tab under System Control and click Begin on the sample list. This starts the automation. • Toggle back to the 212.24 LC pump window and click Start Run on the pumps. This starts the run and the green analysis timer begins in the area to the right of ‘325 MS: ready to collect’ seen in Figure 5. Push the valve button to inject your first sample (water blank) into the LCMS. • Fill the syringe barrel with water and flush out the injection port 2-3 times with a full 25µL each flush. Pull up 10-15µL of the next sample into the syringe, switch the valve to load, and fill the loop with the next sample. Repeat this step as necessary and inject your ubiquitin samples in 2 minute intervals. Figure 5 Figure 6 Figure 7 Figure 8

4. LCMS-325 TQ Operation • Shutdown protocol: Once the analysis completes, stop automation by clicking Stop Automation under the automation tab in System control. Before you go to data work up, activate the method ‘Chem115 MeOH’ and click Start Run in the 212.24 window so the needle and API can be flushed out. • Data Work up: Open your data file by clicking on the data processing tab at the top of the monitor (Figure 9) opening that window (Figure 10). Click on the desired peak and the mass spec data pops up below (Figure 11). Use the arrow keys to locate the highest RIC for the peak of interest. Right click on the mouse to label the peak as desired by choosing New Label for Plot1 (Figure 12). Hover the mouse over the label; when the arrow turns to a ‘hand’ the label and its indicator can be dragged to their desired locations. Print, using the printer icon, choose active chromatogram and spectrum, click the short stack printer icon to send the data to the printer. • Toggle back to the Quad View, click Spray Off button to stop the spray and verify that the spray in the API window has turned to a drip. Allow the drip to continue to flush out the needle and chamber for a few minutes. • Using the Windows tab in System Control, toggle to the 212.24 window and click Stop Pumps. It is now safe to close out System Control, normal exit. Figure 9 Figure 10 Figure 11 Figure 12