Abstract
This chapter presents an integrated and modular microsystem providing rapid analyses of low femtomole of in-gel digests for proteomics applications. Enhancement of sample throughput is facilitated using an autosampler, a microfabricated device comprising a large (2.4-μL total volume) separation channel together with a low-dead-volume interface to nES mass spectrometry. Sample preconcentration is achieved by packing C18 reverse phase or immobilized metal affinity chromatogaphy (IMAC) beads into the large channel of this microfluidic device to adsorb peptides or enrich the sample in phosphopeptides prior to capillary electrophoresis separation and MS detection.
This integrated microfluidic systems enables a sample throughput of up to 12 samples/h with a detection limit of approx 5 nM (25 fmol inj.). Replicate injections of peptide standards indicated that reproducibility of migration time was typically 1.2–1.8%, whereas relative standard deviation (RSD) values of 9.2–11.8% were obtained on peak heights. The application of this device is demonstrated for 2D gel spots obtained from protein extracts of human astrocyte cells and for excised bands of membrane proteins from Neisseria meningitidis. A stepped acetonitrile gradient can be incorporated with the present microfluidic system to enhance selectivity during sample analysis.
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References
S. R. Pennington, M. R. Wilkins, D. F. Hochstrasser, and M. J. Dunn (1997) Proteome analysis: from protein characterization to biological function. Trends in Cell Biol. 7, 168–173
Tyers, M. and Mann, M. (2003) From genomics to proteomics. Nature 422, 193–197.
Rabilloud, T. (2002) Two-dimensional gel electrophoresis in proteomics: still old, old fashion, but still climbs up the mountains. Proteomics 2, 3–10.
Nishihara, J. C. and Champion, K. M. (2002) Quantitative evaluation of proteins in one-and two-dimensional polyacrylamide gels using a fluorescent stain. Electrophoresis 23, 2203–2215.
Gorg, A., Obermaier, C., Boguth, G et al. (2000) The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 21, 1037–1053.
Mann, M. and Wilm, M. (1994) Error tolerant identification of peptide in sequence database by peptide sequence tag. Anal. Chem. 66, 4390–4399.
Eng, J. K., McCormack, A. L., and Yates, J. R. (1994) An approach to correlate MS/MS data to amino acid sequence in a protein database. J. Am. Soc. Mass Spectrom. 5, 976–989.
Perkins, D. N., Pappin, D. J., Creasy, D. M., and Cottrell, J. S. (1999) Probability-based protein identification by searching databases using mass spectrometry data. Electrophoresis 20, 3551–3567.
Link, A. J., Eng, J., Schieltz, D. M., et al. (1999) Direct analysis of protein complexes using mass spectrometry. Nat. Biotechnol. 17, 676–682.
Washburn, M. P., Wolters, and D., Yates III, J. R. (2001) Large scale analysis of the yeast proteome by multidimensional protein identification technology. Nat. Biotechnol. 19, 242–247.
Davis, M. T. and Lee, T. D. (1998) Rapid protein identification using a microscale electrospray LC/MS system on an ion trap mass spectrometer. J. Am. Soc. Mass Spectrom. 9, 194–201.
Moseley A., Blackburn K., Burkhart W., Vissers, H., and Bordoli, R. Proc. 48th ASMS Conference 2000, Long Beach CA.
Xue, Q., Foret, F, Dunayevskiy, Y. M., Zavracky, P. M., McGruer, N. E., and Karger, B. L. (1997) Multichannel microchip electrospray mass spectrometry. Anal. Chem. 69, 426–430.
Ramsey, R. S., and Ramsey, J. M. (1997) Generating electrospray from microchip devices using electroosmotic pumping. Anal. Chem. 69, 1174–1178.
Figeys, D., Ning, Y., and Aebersold, R. (1997) A microfabricated device for rapid protein identification by microelectrospray ion trap mass spectrometry. Anal. Chem. 69, 3153–3160.
Bings, N. H., Wang, C., Skinner, C. D., et al. (1999) Microfluidic devices connected to glass capillaries with minimal dead volume. Anal. Chem. 71, 3292–3296.
Li, J., Wang, C., Kelly, J. F., Harrison, J. D., and Thibault, P. (2000) Rapid and sensitive separation of trace level protein digests using microfabricated device coupled to a high performance quadrupole/time-of-flight mass spectrometer. Electrophoresis 21, 198–210.
Little, D. P., Cornish, T. J., O’Donell, M. J., Braun A., Cotter, R. J., and Koster, H. (1997) MALDI on a chip: analysis of arrays of low-femtomole to subfemtomole quantities of synthetic oligonucleotides and DNA diagnostic products dispensed by a piezoelectric pipet. Anal. Chem. 69, 4540–4546.
Ekstrom, S., Onnerfjord, P., Nilsson, J., Bengtsson, M., Laurell, T., and Marko-Varga, G. (2000) Integrated microanalytical technology enabling rapid and automated protein identification. Anal. Chem. 72, 286–293.
Figeys, D. and Aebersold, R. (1998) Nanoflow solventgradient delivery from a microfabricated device for protein identifications by electrospray ionization mass spectrometry. Anal. Chem. 70, 3721–3727.
Li, J., LeRiche, T., Tremblay, T.-L., et al. (2002) Application of microfluidic devices to Proteomics research: Identification of trace-level protein digests and affinity capture of target peptides. Mol. Cell. Proteomics, 1, 157–168.
Kim, J.-S. and Knapp, D. R. (2001) Miniaturized multichannel electrospray ionization emitters on poly (dimethylsiloxane) microfluidic devices. Electrophoresis 22, 3993–3999.
Bonneil, E., Li, J., Tremblay, T.-L., Bergeron, J. J., and Thibault, P. (2002) Integration of solid phase extraction membranes for sample multiplexing; application to rapid protein identification from gel-isolated protein extracts. Electrophoresis 23, 3589–3598.
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Li, J., Tremblay, TL., Harrison, J., Thibault, P. (2004). Integrated System for Rapid Proteomics Analyses Using Microfluidic Devices Coupled to Nanoelectrospray Mass Spectrometry. In: Strege, M.A., Lagu, A.L. (eds) Capillary Electrophoresis of Proteins and Peptides. Methods in Molecular Biology™, vol 276. Humana Press. https://doi.org/10.1385/1-59259-798-X:305
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DOI: https://doi.org/10.1385/1-59259-798-X:305
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