Multichannel isoelectric focusing devices and high voltage power supplies
Methods, devices, and systems for performing a plurality of isoelectric focusing reactions in parallel are described. In some instances, the disclosed devices may be designed to perform isoelectric focusing or other separation reactions followed by further characterization of the separated analytes using mass spectrometry. The disclosed methods, devices, and systems provide for fast, accurate separation and characterization of protein analyte mixtures or other biological molecules by isoelectric point.
1. A method for performing a plurality of isoelectric focusing separations in parallel, the method comprising:
a) providing a device comprising a planar substrate, wherein the planar substrate comprises a plurality of separation channels;
b) introducing a sample comprising a mixture of analytes into at least two separation channels of the plurality of separation channels;
c) controlling a voltage applied to the at least two separation channels to perform the plurality of isoelectric focusing separations to separate the mixture of analytes of the sample in the at least two separation channels;
d) independently monitoring current of the at least two separation channels to determine a progress of the plurality of isoelectric focusing separations as the plurality of isoelectric focusing separations are performed in parallel;
wherein a first end of the at least two separation channels of the plurality of separation channels is electrically coupled to an anolyte reservoir using a first membrane-containing high voltage electrode fixture;
and wherein the first membrane-containing high voltage electrode fixture comprises: an electrode reservoir;
a membrane;
an inlet fluid channel comprising a first end and a second end; and
an outlet fluid channel comprising a first end that is fluidically coupled to the second end of the inlet fluid channel, and a second end that is fluidically coupled to a separation channel, wherein the inlet fluid channel and the outlet fluid channel intersect with and are fluidically coupled to each other at a plane that defines or is parallel to a surface of the electrode reservoir;
wherein the membrane is disposed within the electrode reservoir at or adjacent to the plane such that the membrane covers all or substantially all of an opening comprising an intersection of the inlet fluid channel and the outlet fluid channel; and
wherein the membrane provides a high hydrodynamic resistance, low electrical resistance connection between a high voltage electrode positioned within the electrode reservoir and a fluid contained within the inlet fluid channel and the outlet fluid channel.
2. The method of claim 1 , wherein a second end of the at least two separation channels of the plurality of separation channels is electrically coupled to a catholyte reservoir using a second membrane-containing high voltage electrode fixture.
3. The method of claim 1 , wherein voltages applied to the at least two separation channels are independently controlled.
4. The method of claim 1 , wherein the sample introduced into each of the at least two separation channels is the same, and a first set of experimental conditions is used to perform the isoelectric focusing separations in a first subset of the at least two separation channels and a second set of experimental conditions is used to perform the isoelectric focusing separations in a second subset of the at least two separation channels.
5. The method of claim 1 , wherein the sample introduced into each of the at least two separation channels is different for at least two subsets of the at least two separation channels and the same set of experimental conditions is used to perform the isoelectric focusing separations in each of the at least two separation channels.
6. The method of claim 1 , further comprising recording a current trace for each of the at least two separation channels while performing the plurality of isoelectric focusing separations.
7. The method of claim 1 , wherein the method further comprises flushing the at least two separation channels following completion of the isoelectric focusing separations and introducing another sample into the at least two separation channels in an automated fashion.
8. The method of claim 1 , wherein detection of a failure in any of the at least two separation channels triggers an automated re-introduction and repeat of the isoelectric focusing separation for the sample that had been introduced into that separation channel.
9. The method of claim 8 , wherein the failure comprises introduction of a bubble, formation of a bubble, an incorrectly prepared sample, an underfilled reagent reservoir, or any combination thereof.
10. The method of claim 9 , wherein the failure is detected by monitoring the current of at least one of the at least two separation channels or by processing an image of at least one of the at least two separation channels.
11. The method of claim 1 , further comprising measuring dynamic light scattering in at least one of the at least two separation channels while performing isoelectric focusing.
12. The method of claim 11 , wherein the measurement of dynamic light scattering provides a determination of a size distribution profile, a determination of an aggregation state, or a determination of a hydrodynamic radius for one or more separated analytes.
13. The method of claim 1 , wherein the membrane is hydrophilic and comprises cellulose or polytetrafluoroethylene (PTFE).
14. The method of claim 1 , wherein the first membrane-containing high voltage electrode fixture further comprises an insert disposed within the electrode reservoir and positioned at or adjacent to the membrane, wherein the insert comprises an inlet fluid path and an outlet fluid path that facilitates substantially bubble-free wetting of a surface of the membrane when the electrode reservoir is filled with an electrolyte solution.