Large-scale Top-down Proteomics Using Capillary Zone Electrophoresis Tandem Mass Spectrometry

This method can help improve large scale and high resolution characterization of protein isoforms and protein post-translational modifications in cells. The main advantage of this technique is that capillary zone electrophoresis mass spectrometry offers highly efficient separation and highly sensitive detection of intact proteins. To pre-treat the capillary, dry it with nitrogen gas at 10 psi for at least 12 hours.

Then, fill the capillary with 50%volume-by-volume 3-propyl methacrylate in methanol using a syringe pump. Seal both ends of the capillary with silica rubber. After incubating at room temperature for at least 24 hours, continue with a linear polyacrylamide coating on the inner wall of the separation capillary, as described in the text protocol.

Burn a portion of the capillary using a gentle flame to remove the polyamide outer coating around four centimeters away from one end of the capillary. Gently clean the burnt portion of the capillary by wiping to remove the polyamide coating completely. Drill a small hole at the end of a 200 microliter tube around the same size as the capillary outer diameter to sufficiently hold the capillary in place once it is threaded through this hole.

Thread the end of the capillary that is close to the burnt portion through the hole until the burnt portion is in the tube. Then, add approximately 150 microliters of HF to the 200 microliter tube so that the HF solution is about halfway up the burnt portion on the capillary. Incubate the capillary in the HF solution at room temperature for 90 to 100 minutes.

Prepare the standard protein mixture and the E.coli sample as described in the text protocol. To set up the capillary zone electrophoresis, or CZE system, load the sample, the background electrolyte, and the separation capillary into CZE auto-sampler. Select sample load in the manual control page of the CZE auto-sampler.

Load the background electrolyte vial into the buffer tray of the auto-sampler, noting its position in the buffer tray. Then, load the sample vial into the sample tray of the auto-sampler, noting its position in the sample tray. Put the auto-sampler to the alignment position using manual control.

Now, load the separation capillary into CZE auto-sampler using the non-etched end of the capillary. Adjust the height of the injection end of the capillary if the sample volume is lower than 50 microliters. Switch the auto-sampler to standby using manual control.

Type the sample position in the system and move the capillary to the sample. Push the injection end of the capillary further down to reach the bottom of the sample vial. Continuing to use the manual control, flush the capillary with the background electrolyte for 20 minutes using a pressure of 20 psi.

Now, mount a commercial electrokinetically pumped sheath flow interface to the front of the mass spectrometer. Fill the sheath buffer reservoir with a buffer containing 10%volume-volume methanol and 0.2%volume-volume formic acid in LCMS-grade water. Flush the T in the interface with the sheath buffer via applying pressure manually with a syringe.

Thread the one millimeter outer diameter end of an electrospray emitter through a sleeve tubing and connect the emitter with one port of the T via a fitting. Simply flush the T manually again with a syringe to fill the emitter with the sheath buffer. Adjust the distance between the orifice of the emitter and the entrance of the mass spectrometer to approximately two millimeters with the help of the camera that came with the interface.

Apply a two to 2.2 kilovolt voltage at the sheath buffer vial for electrospray. Then, adjust the spray voltage to reach a stable electrospray. Apply a low pressure at the injection end of the capillary during this process to make sure there are no bubbles in the capillary.

Turn off the spray voltage and gently thread the etched end of the separation capillary through the T into the emitter until it cannot be pushed further. After stopping the low pressure at the injection end of the capillary, flush the emitter a little bit with sheath buffer. Again, apply two to 2.2 kilovolts of spray voltage to test the spray.

Select new method under the file dropdown menu on the main instrument screen. There, select inlet as starting sample vial, ending sample vial, tray as sample, pressure as five psi, kilovolts as zero, and duration as 95 seconds for a sample injection. Then, set inlet as inlet vial, tray as buffer, pressure as zero psi, kilovolts as 30, and duration as 4, 200 seconds for the separation of the standard protein mixture sample.

For the separation of the E.coli proteome sample, set inlet as inlet vial, tray as buffer, pressure as zero psi, kilovolts as 20, and duration as 6, 600 seconds. Finally, select inlet as inlet vial, tray as buffer, pressure as 10 psi, kilovolts as 30, and duration as 600 seconds for capillary flushing. Now, setup the MS and MS/MS parameters for intact protein analysis using a quadruple ion trap mask spectrometer.

To adjust the tune file settings, turn on intact protein mode and use a trapping pressure of 0.2. Set the ion transfer capillary temperature to 320 degrees Celsius and the S-lens RF level to 55. To perform the CZE MS/MS experiment, start the data acquisition method on the mass spectrometer computer, first by selecting run sequence.

Then, start the capillary electrophoresis sequence on the capillary electrophoresis auto-sampler computer. Perform a database search with TopPIC in the TopPIC suite and in the TopPIC graphical user interface. Click on database file and select an appropriate database for searching.

Click on spectrum file and select the generated ms2. msalign file generated as the input. Select the MS1 feature file and choose the generated feature file.

Set cysteine carbamidomethyl C57 as a fixed modification due to the iodoacetamide treatment. Select the decoy database feature and under cutoff settings, select false discovery rate in the dropdown menu next to spectrum level. Set the false discovery rate to 0.01 at the spectrum level.

Leave generating function unselected and set the error tolerance to 15 parts per million. Set the false discovery rate to 0.05 at the proteoform level. Under advanced parameters, select two for the maximum number of mass shifts in the dropdown menu.

Set the maximum mass shift of unknown modifications to 500 daltons. Leave all other parameters at their default values and click the start button at the bottom right of the graphical user interface. The representative electropherogram for the standard protein mixture is shown.

The standard protein mixture is typically run at least in duplicate to evaluate the separation efficiency and the reproducibility of the system. The separation efficiency can be evaluated with the number of theoretical plates of some proteins. The reproducibility can be evaluated by the relative standard deviations of protein intensity and migration time.

The CZE MS/MS platform can be used for large-scale characterization of proteoforms in various complex proteomes, identifying over 500 proteoforms and 190 proteins from an E.coli proteome in a single run with high confidence. Here, a zoomed in view of the electropherogram can be used to assess the separation window of the system. The very low E-value and spectral FDR suggests the high confidence of the proteoform ID.The high number of matched fragment ions further indicates the high confidence of the ID.While attempting this procedure, it's important to remember to thread the etched separation capillary into the electrospray emitter slowly and gently.

Stable isotope labeling or label-free methods can be incorporated into the CZE MS procedure to determine how proteoform abundance changes in cells across various conditions. After its development, this technique paved the way for researchers in the field of top-down proteomics to explore the roles of proteoforms in regulating various biological processes in cells. Don't forget that working with hydrofluoric acid can be extremely hazardous and precautions such as proper personal protective equipment and having emergency procedures in place should always be taken when performing this procedure.