Q. How much sample do you need?
A. 12 ug. We get at least 3 injections from 12ug so that the sample can be run in triplicate, if needed.
Q. Do you see a difference in pI and peak % between cIEF and Blaze?
A. We designed the system to give the same pI and peak percentage as a traditional IEF system. The Blaze system uses the same ampholytes and pI markers and is able to achieve data comparability to traditional cIEF. We have analyzed over 30 customer samples with equivalent results to their in-house iCIEF data.
Q. You have showed customer’s cIEF data and Blaze cIEF results that are quite consistent. What's the advantage?
A. The advantage of Blaze is iCIEF separation, and UV quantitation with direct peak identification by MS in one assay. Many times, for traditional iCIEF there is an unknown peak and the time to characterize it can be weeks or months. The process involves method development, screening columns, sample scale up, purification, and finally MS analysis. We eliminate all those steps with the 15-min Blaze iCIEF-MS assay.
Q. What is advantage when comparing this tech with ZIP-CHIP?
A. The Blaze system performs imaged CIEF with UV detection and quantitation. Those peaks are mobilized for direct mass spec ID of the iCIEF separation. The ZipChip does a CZE separation and does not do any UV quantitation. The detection is only based on mass spec, which may not be as accurate because of differences in ionization efficiency.
Another advantage of cIEF over CZE is the sensitivity advantage. In cIEF, the entire channel is filled with the sample mixture. During focusing, the sample concentrates about 15X into the discrete peak.
In addition, iCIEF is a standard QC release assay in the biopharmaceutical industry. iCIEF measures the molecules intrinsic pI value.
Q. Can urea be in the system?
A. No, urea is incompatible with the MS analysis. It is not volatile and incompatible with mass spectrometry. We have designed the system to work with MS-friendly reagents and use formamide instead because it is a volatile, MS-compatible chaotrope.
Q. Are you using methylcellulose as additive?
A. We have developed a proprietary hydrophilic coating for the channels in the chip to prevent electroosmotic flow so that methylcellulose use can be eliminated.
Q. After the focusing step, how does mobilization work?
A. Mobilization is the process of transferring the iCIEF-separated peaks to the on-chip electrospray tip for electrospray ionization into the mass spectrometer. After the focusing step, which is typically 5 mins, the voltage circuit is switched between the anolyte and mobilization channels. The peaks are electrophoresed toward the ESI tip and mixed with the mobilizer which reionizes the molecules for detection by MS. The mobilization is also typically 5 mins. The mobilizer is an acidic mixture of 50% ACN and 1% formic acid which is a typical MS condition.
Q. The chip reuse has been a concern in the past with other chip technologies. Have you made improvement in this area?
A. Today the chips are guaranteed to run for 25 injections. We are planning to release a 100-injection chip later in 2021.
Q. Would ampholyte signal interfere with smaller protein subunits (chains) that would run at a lower m/z scale on the mass spectrometer?
A. We have not seen signal suppression of the protein signal in the MS. We are separating some of the ampholytes away from the MS during mobilization by setting the voltage so they migrate toward the mobilizer channel, rather than to the MS.
Q. When will the instrument be available?
A. We have been taking orders since June and will be shipping systems at the end of 2020.
Q. Which MS system is the Blaze iCIEF-MS system compatible with?
A. The Blaze system is compatible with the Thermo Q-Exactive, Q-Exactive Plus, Exactive EMR, and SCIEX TripleTOF 6600 and 6600+. We are adding compatibility with other vendors’ mass spectrometers in 2021. Reach out to us if you would like to discuss compatibility with your mass spec!