The overall goal of this procedure is to demonstrate how capillary electrophoresis-mass spectrometry, or CE-MS, using a sheathless, porous-tip interface, can be used for the analysis of highly polar and charged metabolites. Assuming no metabolites can be often charged, and they can be cationic or anionic, and to separate them electrolitic separations like capillary electrophoresis, is the most suitable method. However, in recent years, the sensitivity was not good enough.
That was due to the interfacing and the method was also not robust. But I think we nicely solved both problems using a sheath as free interface and to use this new protocol. Capillary electrophoresis-mass spectrometry using a novel sheathless porous tip interface can be used for addressing key clinical questions in the field of metabolomics.
For example, the profiling of highly polar charged metabolites can be easily done with this technology. S composites separated on base of charge to size ratio. More often this technique is very suited for the profiling of this type of compounds volume labeled to biological samples.
A unique feature of the proposed sheathless CE-MS methodology is that they can be used for the profiling of anionic and cationic metabolites by only switching the CE voltage polarity and the MS detection polarity. Generally people new to this method will struggle because CE-MS is considered as a relatively new and complicated technique. To begin this procedure, place a new bare fused silica cartridge with a porous tip emitter in the capillary zone electrophoresis, or CE instrument.
Apply a forward methanol rinse at 50 psi for 15 minutes with the software controlling the CE instrument. And visually check whether liquid is flowing out from the capillary outlet. Then, perform a rinse in the opposite direction at 50 psi for five minutes using the background electrolite, or BGE solution, to visually examine whether liquid is flowing out from the conductive capillary.
Repeat the previous step at a pressure of 100 psi in case liquid has not been observed flowing out of the capillary outlet. Next, rinse the separation capillary with water at 50 psi for 10 minutes, followed by 0.1 molar sodium hydroxide at 50 psi for 10 minutes, water at 50 psi for 10 minutes, and BGE solution at 50 psi for 10 minutes. Following this, remove the sprayer tip of the fused silica cartridge from the water tube and install it in the nanospray source adapter for coupling to the MS instrument.
Ensure that the height of the BGE solution vials in the CE instrument match the height of the sprayer tip. Check for flow of liquid through the conductive capillary by rinsing with BGE solution at 50 psi for five minutes. During this rinsing step, a liquid drop at the base of the electrospray ionization, or ESI sprayer needle, should be observed.
After rinsing, flush the separation capillary with BGE solution at 50 psi for 10 minutes in the forward direction. A liquid drop should be observed at the tip of the porous tip emitter during this step. Now, position the porous tip emitter to the entrance of the MS inlet at an approximate distance of two to three mm.
Apply a voltage of 30 kV using a ramp time of one minute and start acquiring MS data in the mass-to-charge range from 65 to 1000 for metabolic profiling studies using first an ESI voltage of zero. Set the ESI voltage to 1000 V while measuring the data. Increase the ESI voltage with increments of 200 V until a constant background signal is observed for at least 15 minutes.
Transfer 20 microliters of a previously prepared anionic metabolite standard mixture into an empty 100 microliter microvial. Place the microvial in a CE vial, and place the CE vial in the inlet sample tray. Start a previously created MS acquisition negative ion mode method, and subsequently start the CE sequence using the software controlling the CE instrument.
Following this, rinse the separation capillary with BGE solution at 50 psi for three minutes, followed by injections at two psi for 60 seconds and one psi for 10 seconds. Apply a voltage of 30 kV with a ramp time of one minute and a pressure of 0.5 psi for 30 minutes at the inlet. After a 30 minute electophoretic separation, stop MS data acquisition and decrease the CE voltage to 1 kV using a ramp time of five minutes.
Between sample injections, rinse the separation capillary with water, 0.1 molar sodium hydroxide, water, and BGE solution at 30 psi for three minutes. Once the runs are complete, analyze the recorded data by determining the migration times and the signal intensity of the analyzed anionic metabolite mixture. Assess whether the anionic metabolite standards appear in the region between 10 and 28 minutes.
Then, check whether the three structurally related isomers, D-glucose 1-phosphate, D-glucose 6-phosphate, and D-fructose 6-phosphate, are partially separated. The separation performance of the sheathless CE-MS method for the analysis of highly polar anionic metabolites is demonstrated for three structurally related sugar phosphate isomers. Though a baseline separation was not obtained for these three analytes, a partial separation is sufficient to allow their selective detection by MS, as these analytes have the same exact mass.
When analyzing biological samples, with this sheathless CE-MS methodology, it is important to regularly check the analytical performance over time using academic standards. Overall, the sheathless methodology allows the profiling of these type of compounds in ultra small biological samples, thereby opening up a lot of possibilities for analyzing these type of samples in the biomedical sciences fields, bioanalytical chemistry fields, and research areas outside. After watching this video, you should have a good understanding of how to couple CE to MS via a sheathless porous interface for the profiling of highly polar and charged metabolites.
