The overall goal of the HS-MRM assay is to reduce the MRM interference phenomenon which affects peptide or protein quantification by combining ion mobility separation of peptide precursors with high-resolution mass spectrometric detection of fragment ions. This method can help answer key questions in the area of bio-therapeutic characterization as well as protein or peptide biomarker quantification and quantitative proteomics. The main advantage of this technique is that it allows detection of low-abundant host cell proteins that might be present in bio-pharmaceutical proteins.

First, click on create analysis method in the data acquisition software and choose generate and acquire and process method. Type the method name and description, browse to the method folder and click next. For analysis type, choose peptide map IMS and click next.

Then select the current system configuration and click next and finish. In the newly created method, click on the instruments tab, select the binary solvent manager, and edit the gradient settings to achieve peptide separation at a flow rate of 0.2 milliliters per minute. Use a gradient dilution from one to 40%solvent B in 30 minutes followed by a two-minute column wash and a 10-minute equilibration with a total runtime of 45 minutes.

Next, select the sample manager and set the sample temperature to 10 degrees Celsius and the column temperature to 60 degrees Celsius. In the instruments tab, select the Vion IMS QTof instrument. In the settings section, select sensitivity for analyzer mode and enter three kilovolts for capillary voltage, 100 degrees Celsius for source temperature, 250 degrees Celsius for desolvation temperature, 50 liters per hour for cone gas flow, and 500 liters per hour for desolvation gas flow.

Click on the experiment tab and select high definition MSE for mode. Then enter 100 for low mass, 2000 for high mass, and 0.4 seconds for scan time. In the collision energy section, type six electron volts for the low energy setting and choose a high energy ramp from 15 to 40 electron volts.

For the acquisition time, use a custom runtime between five and 30 minutes. Next, click on the options tab and select the automatic lock correction mode with an automatic lock correction interval of five minutes. Then select the continue with lock correction option and to make sure that the automatic detector check is disabled.

In the current system configuration menu, select the Vion IMS QTof instrument and click on tools. Enter three kilovolts for capillary voltage, 40 volts for sampling cone, 100 volts for source offset, 100 degrees Celsius for source temperature, 250 degrees Celsius for desolvation temperature, 50 liters per hour for cone gas flow, 500 liters per hour for desolvation gas flow, and three kilovolts for reference capillary voltage. Analyze a previously prepared 10 nanomolar faux spiked sample using the LC-HDMSE assay just described by injecting 10 microliters of sample into the instrument.

The individual sequences of six phosphorylase B peptide standards contained in the faux peptide mixture are shown here along with their retention times and their most abundant precursors observed in the HDMSE experiment. The HDMSE spectra acquired for one of the faux peptides spiked in the Infliximab digest are displayed here. In the data acquisition software, click on create analysis method and choose generate and acquire and process method.

Type the method name and description, browse to the method folder and click next. For analysis type, choose quantify, select Quantify Assay Tof 2D Chromatographic and click next. Then select the current system configuration and click next and finish.

Click on the instruments tab in the newly created method. Select the binary solvent manager and edit the gradient settings to achieve peptide separation at a flow rate of 0.2 milliliters per minute. Use gradient dilution from one to 40%solvent B in 30 minutes followed by a two-minute column wash and a 10-minute equilibration with a total runtime of 45 minutes.

In the instruments tab, select the sample manager and set the sample temperature to 10 degrees Celsius and the column temperature to 60 degrees Celsius. Now select the Vion IMS QTof instrument and in the settings section, select sensitivity for analyzer mode. Then enter three kilovolts for capillary voltage, 100 degrees Celsius for source temperature, 250 degrees Celsius for desolvation temperature, 50 liters per hour for cone gas flow, and 500 liters per hour for desolvation gas flow.

Click on the experiment tab and for experiment type, choose function table one or more MS, MSMS, or MRM functions. Delete the default acquisition function, click on add plus MRM, and edit the retention window parameters in the runtime section. In the Tof MRM function editor, enter the peptide precursor master charge ratio, select low for precursor resolution, and enter the precursor charge state.

Then input the product master charge ratio 16 electron volts for the starting collision energy, 0.1 seconds for the fixed scan time, and wide for the spectrum column. Using the duplicate button, add 10 more MRM transitions and use different collision energy values in the range of 18 to 36 electron volts. After establishing the three best transitions for each peptide, a Tof MRM experiment was performed to find the optimum collision energy to maximize the signals generated for each peptide.

Next, set up an HS-MRM method containing only one fragment ion per peptide. Modify the previously created Tof MRM method by selecting the HS-MRM function instead of the Tof MRM function. In the HS-MRM function editor, enter the master charge ratio, select low for precursor resolution, and enter the precursor charge state.

Then input the precursor CCS value and select low for precursor CCS resolution. For the product ion, enter its master charge ratio, select the optimum collision energy, choose a scan time of 0.4 seconds, and select a wide spectrum setting to include all of its isotopes. The final HS-MRM assay retains only the best transition for each peptide and is used for analyzing all spiked samples.

In the analysis method created for the HS-MRM data acquisition method, click on the purpose tab and choose manage components. From the dropdown menu under experiment type, select the HS MSMS/HS MRM option. Click on create and enter the component name, expected retention time, extraction time window, and precursor master charge ratio.

Select XIC extraction mode and type the expected CCS value. For the fragment ion, enter its charge state and master charge ratio. Then enter all peptide components monitored by the HS-MRM assay.

In the purpose tab of the same HS-MRM acquisition method, click on default amounts and enter 0.1, one, 10, and 100 nanomolar for all faux peptide components. After entering the first value, right click and select the fill down option to enter the same concentration for all components. Repeat the same procedure for all four concentrations of the calibration curve.

Now move to the processing tab and click on extraction settings. Input 10 PPM for mass tolerance. Then click on CCS drift time tolerance and input 5%for CCS tolerance.

In the processing tab, select the peak processing settings. In the smooth section, select mean as the smoothing algorithm, one as the half width, and one as the iterations. In the integrate section, make sure that the automatic peak width and automatic detection options are both checked.

Next, select the targeted screen settings in the processing tab and select the option of closest to RT in the RT window. Then input 0.2 minutes for the retention time tolerance. Select quantitation settings and in the calibrate menu, choose linear for calibration curve fit type, one over x squared for weight type, concentration for component value type, millimoles per liter for component value units, and none for compute calibration points by averaging.

In the quantify menu, select area as the response value. Examples of HS-MRM chromatograms generated for the faux peptides across all the concentrations are presented here. The calibration curves obtained for each peptide following the integration of HS-MRM peaks are displayed here.

The peak area relative standard deviation calculated based on four replicate injections is summarized here. Because the high selectivity MRM assay incorporates both ion mobility precursor separation and targeted high resolution mass spectrometric detection, it has great potential for becoming a fast, high-throughput monitoring assay for multiple host-cell proteins across batches of biopharmaceuticals. After watching this video, you should have a good understanding of how to set up a high selectivity MRM assay on a Vion IMS QTof instrument.