Devices and methods for sample characterization
Devices and methods for characterization of analyte mixtures are provided. Some methods described herein include performing enrichment steps on a device before expelling enriched analyte fractions from the device for subsequent analysis. Also included are devices for performing these enrichment steps.
1. A method, comprising:
(a) applying a first electric field across a first fluid channel in a microfluidic device to separate a mixture of analytes and ampholytes via isoelectric focusing while imaging the first fluid channel or a portion thereof;
(b) applying a second electric field across the first fluid channel to mobilize the separated analytes while imaging the first fluid channel or a portion thereof;
(c) expelling the mobilized analytes via electrospray ionization from an orifice in the microfluidic device into a mass spectrometer; and
(d) correlating separated analyte peaks detected in (a) by the imaging of the first fluid channel or a portion thereof with mass spectrometer data for the separated analytes.
2. The method of claim 1 , wherein the mixture of analytes comprises a mixture of intact proteins.
3. The method of claim 1 , wherein the microfluidic device comprises an optical slit that provides optical access to the first fluid channel, and wherein the imaging comprises detecting light that has passed through or is emitted from the first fluid channel.
4. The method of claim 1 , wherein the imaging comprises absorbance imaging to detect and monitor separated analyte peaks.
5. The method of claim 1 , wherein the imaging comprises fluorescence imaging to detect and monitor separated analyte peaks.
6. The method of claim 5 , wherein the fluorescence imaging comprises imaging of native fluorescence.
7. The method of claim 1 , further comprising introducing isoelectric point (pI) markers into the first fluid channel prior to performing the isoelectric focusing in (a), and wherein the imaging further comprises detection and monitoring of positions of the pI markers.
8. The method of claim 7 , wherein the positions of the pI markers are used to determine an isoelectric point (pI) for one or more separated analytes.
9. The method of claim 1 , wherein the microfluidic device further comprises a nebulizing gas delivery channel for facilitating the electrospray ionization.
10. The method of claim 1 , wherein the microfluidic device further comprises a second fluid channel that is in fluid communication with an end of the first fluid channel that is opposite an end that is in fluid communication with the orifice.
11. The method of claim 10 , wherein a chromatographic enrichment is performed in the second fluid channel prior to performing the isoelectric focusing separation in (a).
12. The method of claim 11 , wherein the chromatographic enrichment comprises a reversed-phase, immunoprecipitation, ion exchange, size exclusion, or affinity chromatographic enrichment.
13. A method, comprising:
(a) separating a mixture of analytes in a first fluid channel in a microfluidic device while imaging the first fluid channel or a portion thereof;
(b) mobilizing the separated analytes in the first fluid channel while imaging the first fluid channel or a portion thereof;
(c) expelling the mobilized analytes via electrospray ionization from an orifice in the microfluidic device into a mass spectrometer; and
(d) correlating separated analyte peaks detected in (a) by the imaging of the first fluid channel or a portion thereof with mass spectrometer data for the separated analytes.
14. The method of claim 13 , wherein the separation in (a) is performed using isoelectric focusing or capillary electrophoresis.
15. The method of claim 13 , wherein the separation in (a) is performed using chromatography.
16. The method of claim 13 , wherein the analyte mixture comprises proteins.
17. The method of claim 16 , wherein the proteins comprise intact proteins.
18. The method of claim 13 , wherein the imaging comprises absorbance imaging to detect and monitor separated analyte peaks.
19. The method of claim 13 , wherein the imaging comprises fluorescence imaging to detect and monitor separated analyte peaks.
20. The method of claim 19 , wherein the fluorescence imaging comprises imaging of native fluorescence.
21. The method of claim 13 , wherein the mobilization of separated analytes in (b) is performed by introducing an eluent into the first fluid channel using pressure.
22. The method of claim 13 , wherein the mobilization of separated analytes in (b) comprises chemical mobilization or electrokinetic mobilization.
23. The method of claim 13 , wherein the microfluidic device comprises an optical slit that provides optical access to the first fluid channel, and wherein the imaging comprises detecting light that has passed through or is emitted from the first fluid channel.
24. The method of claim 13 , further comprising introducing isoelectric point (pI) markers into the first fluid channel prior to performing the isoelectric focusing in (a), and wherein the imaging further comprises detection and monitoring of positions of the pI markers.
25. The method of claim 24 , wherein the positions of the pI markers are used to determine an isoelectric point (pI) for one or more of the separated analytes.
26. The method of claim 13 , wherein the microfluidic device further comprises a second fluid channel that is in fluid communication with an end of the first fluid channel that is opposite an end of the first fluid channel that is in fluid communication with the orifice, and wherein a chromatographic enrichment is performed in the second fluid channel prior to performing the separation in (a).
27. The method of claim 16 , wherein the chromatographic enrichment comprises a reversed-phase, immunoprecipitation, ion exchange, size exclusion, or affinity chromatographic enrichment.
28. The method of claim 13 , wherein the microfluidic device further comprises a nebulizing gas delivery channel for facilitating the electrospray ionization.