Summary
HPLC-chip/MS is a novel nanoflow analytical technology conducted on a microfabricated chip that allows for highly efficient HPLC separation and superior sensitive MS detection of complex proteomic mixtures. This is possible through on-chip preconcentration and separation with fluidic connection made automatically in a leak-tight fashion. Minimum precolumn and postcolumn peak dispersion and uncompromised ease of use result in compounds eluting in bands of only a few nanoliters. The chip is fabricated out of bio-inert polyimide-containing channels and integrated chip structures, such as an electrospray emitter, columns, and frits manufactured by laser ablation technology. Meanwhile, a variety of HPLC-chips differing in design and stationary phase are commercially available, which provide a comprehensive solution for applications in proteomics, glycomics, biomarker, and pharmaceutical discovery. The HPLC-chip can also be easily integrated into a multidimensional separation workflow where different orthogonal separation techniques are combined to solve a highly complex separation problems. In this chapter, we describe in detail the methodological chip usage and functionality and its application in the elucidation of the protein profile of human nucleoli.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Issaq, J. H., Chan, K. C., Janini, G. M., Conrads, T. P., and Veenstra, T. D. (2005). Multidimensional separation of peptides for effective proteomic analysis. J. Chromatogr. B 817, 35–47
Jandera, P. (2006). Column selectivity for two-dimensional liquid chromatography. J. Sep. Sci. 29, 1763–1783
Wolters, D. A., Washburn M. P., and Yates, J. R. (2006). An automated multidimensional protein identification technology for shotgun proteomics. Anal. Chem. 73, 5683–5690
Wilm, M. and Mann M. (1996). Analytical properties of the nanoelectrospray ion source. Anal Chem., 68, 1–8
Koster, S. and Verpoorte, E. (2007). A decade of microfluidic analysis coupled with electrospray mass spectrometry: an overview. Lab Chip, 7, 1394–1412
Yin, H., Killeen, K., Brennen, R., Sobek, D., Werlich, M., and van de Goor T. (2005). Microfluidic chip for peptide analysis with an integrated HPLC column, sample enrichment column and nanoelectrospray tip. Anal. Chem. 77, 527–533
Yin, H. and Killeen, K. (2007). The fundamental aspects and applications of Agilent HPLC-Chip. J. Sep. Sci. 30, 1427–1434
Fortier, M.-H., Bonneil, E., Goodley, P., and Thibault, P. (2005). Integrated microfluidic device for mass spectrometry-based proteomics and its application to biomarker discovery programs. Anal. Chem. 77, 1631–1640
Hardouin, J., Duchateau, M., Caron-Joubert, R., and Caron, M. (2006). Usefulness of an integrated microfluidic device (HPLC-Chip-MS) to enhance confidence in protein identification by proteomics. Rapid Commun. Mass Spectrom. 20, 3236–3244
Ninonuevo, M. R., Park, Y., Yin, H., Zhang, J., Ward, R. E., Clowers, B. H. et al (2006). A strategy for annotating the human milk glycome. J. Agric. Food Chem. 54, 7471–7480
Vollmer, M., Hoerth, P., Rozing, G., Coute, Y., Grimm, R., Hochstrasser, D., and Sanchez, J.-C. (2006). Multi-dimensional HPLC/MS of the nucleolar proteome using HPLC-chip/MS. J. Sep. Sci. 29, 499–509
Hoerth, P., Miller, C.A., Preckel, T., and Wenz, C. (2006). Efficient fractionation and improved protein identification by peptide OFFGEL electrophoresis. Mol. Cell. Proteomics 5.10, 1968–1974
Staes, A., Timmerman, E., Van Damme, J., Helsens, K., Vandekerckhove, J., Vollmer, M., and Gevaert, K. (2007). Assessing a novel microfluidic interface for shotgun proteome analyses. J. Sep. Sci. 30, 1468–1476
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254
Giddings, J. C. (1987). Concepts and comparisons in multidimensional chromatography. J. High Res. Chromatogr. 10, 319–323
Zolotarjova, N., Martosella, J., Nicol, B., Bayley J.,. and Boyes, B. (2005). Differences among techniques for high-abundant protein removal depletion. Proteomics 5, 3004–3013
Scherl, A., Coute, Y., Deon, C., Calle, A., Karine, K., Sanchez, J.-C. et al (2002). Functional proteomic analysis of human nucleolus. Mol. Biol. Cell. 13, 4100–4109
Madjar, J.-J., Arpin, M., Buisson, M., Reboud, J. P. (1979). Spot position of rat ribosomal proteins by four different two-dimensional electrophoreses in polyacrylamide gel. Mol. Gen. Genet. 171, 121–134
Lippincott, J. and Apostol I. (1999). Carbamylation of cysteine: a potential artifact in peptide mapping of hemoglobins in the presence of urea. Anal. Biochem. 267, 57–64
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Vollmer, M., van de Goor, T. (2009). HPLC-Chip/MS Technology in Proteomic Profiling. In: Foote, R., Lee, J. (eds) Micro and Nano Technologies in Bioanalysis. Methods in Molecular Biology™, vol 544. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-483-4_1
Download citation
DOI: https://doi.org/10.1007/978-1-59745-483-4_1
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-934115-40-4
Online ISBN: 978-1-59745-483-4
eBook Packages: Springer Protocols