Abstract
Peptide study and analysis widely involve liquid chromatography. Among the different strategies available, reversed-phase liquid chromatography (RP-HPLC) is one of the methods of choice to separate species in a nontargeted approach. The compounds are sorted according to their hydrophobicity, even though the experimental order of elution could change according to the nature of the mobile phase and the stationary phase. In our work, we have developed protocols to resolve hundred of peptidic species. To overcome the limitations of peak capacity of RP-HPLC alone, it has been coupled downstream to tandem mass spectrometry using two different ionization modes. To overcome the limitations of peak capacity of RP-HPLC MS/MS, it has been coupled upstream to strong cation exchange liquid chromatography. Multidimensional analysis allows for a deeper description of a sample because the limit of detection is often due to a lack of dynamic range of the detection itself rather than due to a lack of sensitivity. In this chapter, different protocols are presented. They should be considered as examples that could be used as starting point for new protocols optimization. Even if RP-HPLC is a universal peptide separation method, it should be optimized according to the specific characteristics of the peptide(s) of interest.
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
References
Perry, M., Li, Q., and Kennedy, R.T. (2009) Review of recent advances in analytical techniques for the determination of neurotransmitters. Analytica Chimica Acta 653, 1–22.
Fricker, L.D., Lim, J., Pan, H., and Che, F.Y. (2006) Peptidomics: identification and quantification of endogenous peptides in neuroendocrine tissues. Mass. Spec. Rev. 25, 327–344.
Saz, J.M., and Marina, M.L. (2008) Application of micro- and nano-HPLC to the determination and characterization of bioactive and biomarker peptides. J. Sep. Sci. 31, 446–458.
Boonen, K., Landuyt, B., Baggerman G., et al (2008) Peptidomics: The integrated approach of MS, hyphenated techniques and bioinformatics for neuropeptide analysis. J. Sep. Sci. 31, 427–445.
Boonen, K., Husson, S.J., Landuyt, B., et al (2010) Identification and relative quantification of neuropeptides from the endocrine tissues. In: Soloviev, M. (ed.), Peptidomics, Meth. Mol. Biol. 615 Humana Press, Springer Science.
Pesek, J.J., Matyska, M.T., and Pindi Venkat, J. (2008) Evaluation of protein, peptide, and amino acid retention on C5 hydride-based stationary phases. J. Sep. Sci. 31, 2560–2566.
Wujcik, C.E., Tweed, J., and Kadar, E.P. (2010) Application of hydrophilic interaction chromatography retention coefficients for predicting peptide elution with TFA and methanesulfonic acid ion-pairing reagents. J. Sep. Sci. 33, 826–833.
Li, Y., and Lee, M.L. (2009) Biocompatible polymeric monoliths for protein and peptide separations. J. Sep. Sci. 32, 3369–3378.
Donato, P., Dugo, P., Cacciola, F., et al (2009) High peak capacity separation of peptides through the serial connection of LC shell-packed columns. J. Sep. Sci. 32, 1129–1136.
Machtejevas, E., Marko-Varga, G., Lindberg,C. et al (2009) Profiling of endogenous peptides by multidimensional liquid chromatography: On-line automated sample cleanup for biomarker discovery in human urine, J. Sep. Sci. 32, 2223–2232.
Storme, M.L., t’Kindt, R.S., and Van Bocxlaer, J.F. (2009) Improved analyte detectability of proteins and peptide lysates by means of multiple large-volume injection in LC-MS. J. Sep. Sci. 32, 2346–2352.
Hesse, A.M., Marcelo, P., Rossier, J. et al (2008) Simple and universal tool to remove on-line impurities in mono- or two-dimensional liquid chromatography-mass spectrometry analysis. J. Chromatogr. A 1189, 175–182.
Kraut, A., Marcellin, M., Adrait, A. et al. (2009) Peptide storage: are you getting the best return on your investment? Defining optimal storage conditions for proteomics samples. J. Proteome Res. 8, 3778–3785.
Acknowledgments
The authors would like to thank Emmanuelle Demey and Iman Haddad for technical assistance. The 2D-LC work was part of A.M. Hesse’s PhD work, which was cofinanced by the CNRS (Centre National de la Recherche Scientifique) and by L’Oréal. The nano-LC-MALDI work was part of S. Ndiaye’s PhD work, which was financed by UPMC Paris 6. The hardware configuration was financed by the town of Paris (ESPCI ParisTech) for the dual LC configuration and by the Réseau National des Genopoles (RNG) for the monodimensional LC configurations.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Hesse, AM., Ndiaye, S., Vinh, J. (2011). Reversed-Phase HPLC and Hyphenated Analytical Strategies for Peptidomics. In: Merighi, A. (eds) Neuropeptides. Methods in Molecular Biology, vol 789. Humana Press. https://doi.org/10.1007/978-1-61779-310-3_13
Download citation
DOI: https://doi.org/10.1007/978-1-61779-310-3_13
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-309-7
Online ISBN: 978-1-61779-310-3
eBook Packages: Springer Protocols