Abstract :
[en] Introduction:
Sheathless CE-MS interfaces allow increase in sensitivity by coupling low-flow electrospray ionization and tandem MS detection. Peak intensity will depend on spray voltage as well as migration and injection conditions. Nevertheless, these parameters influence each other and require methodical optimization to get the most of each instrument. In the present work we share our experience with sheathless CE-MS and neutral coating to analyze peptide and protein samples.
Methods:
Experiments were conducted on a CESI 8000 capillary electrophoresis holding a neutral coated OptiMS cartridge and coupled to a QT 6500 mass spectrometer. Separation buffer and voltage, curtain gas and source temperature were conserved through experiments. Separation pressure and source voltage were optimized while applying voltage and pressure on separation buffer spiked with the peptide used (pI 9.5 marker from the Advance cIEF starter kit by Sciex). A daily reference run was used to compare modifications to the injection despite variable capillary performance. Finally, shifts in spray voltage due to injection parameters were determined using sequences of runs with different spray voltages.
Preliminary results:
Decreasing separation pressure from 5 to 1.5 psi increased peptide intensity; electrokinetic injection (EKI) increased peak intensity compared to hydrodynamic injection (HDI); the HDI of a water plug before the EKI increased peak intensity further, as well as a high percentage of acetonitrile in the sample medium. Finally, we compared our initial and our final conditions. In both cases, a positive Q1 scan of 1000 Da/s for m/z 300 to 1000 was acquired, and the electropherograms display the extracted ion current for a 1 m/z interval centered on the m/z of the doubly charged peptide.
In the initial method, the peptide was diluted in BGE and was introduced by HDI (1 % of total length); 5 psi pressure were applied to both inlet and outlet; source voltage was 1800 V. When analyzing a 1:160 (v/v) dilution of the peptide, the intensity recorded for [M+2H]2+ was 9.3e7 counts.
In the final method, the peptide was diluted in 75:25 acetonitrile:water (v/v) and was introduced by EKI (+ 10 kV 100s). Before the EKI, a HDI of water (0.5 % of total length) was performed, and after the EKI, separation buffer was introduced by HDI (0.5 % of total length). The separation pressure was changed to 1.5 psi and the spray voltage adjusted to 1600 V. When analyzing a 1:160000 (v/v) dilution of the peptide, the recorded intensity of [M+2H]2+ was 9e8 counts. Therefore, following these guidelines, we were able to increase intensity by a 10000 factor.
Novel aspect:
Frequent monitoring of spray voltage and peak intensity in similar conditions allows good inter-run comparison and troubleshooting.
