Abstract
Body fluids and body tissues have a myriad of peptides and proteins that, very often, the traditional methodologies of proteomics, such as conventional gel electrophoresis or mass spectrometry, are unable to characterize. We describe two protocols to characterize high molecular weight peptides (>3 kDa) and intact proteins involving on-line trypsin digestion, separation of the digests by nano-HPLC, and analysis by mass spectrometry using two different ionization sources (matrix-assisted laser desorption and electrospray ionization). These protocols have the advantage of promoting protein denaturation in an aqueous-organic solvent, which reduces the derivatization of the sample and facilitates an in-depth analysis for detection and identification of proteins. Additional advantages include the following: (1) integration of these protocols into standard proteomic workflows after the preprocessing of samples and separation; (2) use of high-resolution monolithic columns; and (3) the ability to acquire information from minimal amounts of sample.
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
Hou, W., Ethier, M., Smith, J.C., et al. (2007) Multiplexed proteomic reactor for the processing of proteomic samples. Anal Chem 79, 39–44.
Ferguson, P.L., Smith, R.D. (2003) Proteome analysis by mass spectrometry. Annu Rev Biophys Biomol Struct 32, 399–424.
Roe, M.R., Griffin, T.J. (2006) Gel-free mass spectrometry-based high throughput proteomics: tools for studying biological response of proteins and proteomes. Proteomics 6, 4678–4687.
Gorg, A., Weiss, W., Dunn, M.J. (2004) Current two-dimensional electrophoresis technology for proteomics. Proteomics 4, 3665–3685.
Martosella, J., Zolotarjova, N., Liu, H., et al. (2005) Reversed-phase high-performance liquid chromatographic prefractionation of immunodepleted human serum proteins to enhance mass spectrometry identification of lower-abundant proteins. J Proteome Res 4, 1522–1537.
Martosella, J., Zolotarjova, N., Liu, H., et al. (2006) High recovery HPLC separation of lipid rafts for membrane proteome analysis. J Proteome Res 5, 1301–1312.
Mant, C.T., Hodges, R.S. (2008) Mixed-mode hydrophilic interaction/cation-exchange chromatography (HILIC/CEX) of peptides and proteins. J Sep Sci 31, 2754–2773.
Boersema, P.J., Divecha, N., Heck, A.J., et al., (2007) Evaluation and optimization of ZIC-HILIC-RP as an alternative MudPIT strategy. J Proteome Res 6, 937–946.
Breuker, K., Jin, M., Han, X., et al., (2008) Top-down identification and characterization of biomolecules by mass spectrometry. J Am Soc Mass Spectrom 19, 1045–1053.
Freije, J.R., Mulder, P.P., Werkman, W., et al. (2005) Chemically modified, immobilized trypsin reactor with improved digestion efficiency. J Proteome Res 4, 1805–1813.
Peterson, D.S., Rohr, T., Svec, F., et al. (2002) High-throughput peptide mass mapping using a microdevice containing trypsin immobilized on a porous polymer monolith coupled to MALDI TOF and ESI TOF mass spectrometers. J Proteome Res 1, 563–568.
Hedstrom, M., Grey, C.E., Gáspár, S., et al. (2007) Miniaturized on-line digestion system for the sequential identification and characterization of protein analytes. J Chromatogr A 1146, 17–22.
Lee, J., Soper, S.A., Murray, K.K. (2009) Development of an efficient on-chip digestion system for protein analysis using MALDI-TOF MS. Analyst 134, 2426–2433.
Massolini, G., Calleri, E. (2005) Immobilized trypsin systems coupled on-line to separation methods: recent developments and analytical applications. J Sep Sci 28, 7–21.
Graumann, J., Dunipace, L.A., Seol, J.H., et al. (2004) Applicability of tandem affinity purification MudPIT to pathway proteomics in yeast. Mol Cell Proteomics 3, 226–237.
Zhou, W., Capello, M., Fredolini, C., et al. (2010) Mass spectrometry analysis of the post-translational modifications of alpha-enolase from pancreatic ductal adenocarcinoma cells. J Proteome Re 9, 2929–2936.
Kato, M., Sakai-Kato, K., Jin, H., et al. (2004) Integration of on-line protein digestion, peptide separation, and protein identification using pepsin-coated photopolymerized sol-gel columns and capillary electrophoresis/mass spectrometry. Anal Chem 76, 1896–1902.
Stigter, E.C., de Jong, G.J., van Bennekom W.P. (2008) Pepsin immobilized in dextran-modified fused-silica capillaries for on-line protein digestion and peptide mapping. Anal Chim Acta 619, 231–238.
Ye, M., Hu, S., Schoenherr, R.M., et al. (2004) On-line protein digestion and peptide mapping by capillary electrophoresis with post-column labeling for laser-induced fluorescence detection. Electrophoresis 25, 1319–1326.
Zheng, S., Yoo, C., Delmotte, N., et al. (2006) Monolithic column HPLC separation of intact proteins analyzed by LC-MALDI using on-plate digestion: An approach to integrate protein separation and identification. Anal Chem 78, 5198–51204.
Vitorino, R., Guedes, S., Tomer, K., et al. (2008) On-plate digestion using a commercial microfraction collector for nano-HPLC matrix-assisted laser desorption/ionization tandem time-of-flight protein analysis. Anal Biochem 380, 128–130.
Noga, M., Sucharski, F., Suder, P., et al. (2007) A practical guide to nano-LC troubleshooting. J Sep Sci 30, 2179–2189.
Krenkova, J., Kleparnik, K., Foret, F. (2007) Capillary electrophoresis mass spectrometry coupling with immobilized enzyme electrospray capillaries. J Chromatogr A 1159, 110–118.
Creasy, D.M., Cottrell, J.S. (2004) Unimod: Protein modifications for mass spectrometry. Proteomics 4, 1534–156.
Garavelli, J.S., (1999) The RESID Database of protein structure modifications. Nucleic Acids Res 27, 198–199.
Vitorino, R., Calheiros-Lobo, M.J., Duarte, J.A., et al. (2008) Peptide profile of human acquired enamel pellicle using MALDI tandem MS. J Sep Sci 31, 523–537.
Getie-Kebtie, M., Franke, P., Aksamit, R., et al. (2008) Experimental evaluation of protein identification by an LC/MALDI/on-target digestion approach. J Proteome Res 7, 3697–3707.
Acknowledgments
The authors wish to acknowledge their appreciation for the financial support provided by the “Fundação para a Ciência e Tecnologia” (FCT – Grant no. PTDC/QUI/72683/2006). Czech grants MEYS CR – LC06035, GACR P301/11/2055, P206/11/P004 and Institutional research plan AV0Z 40310501 are also acknowledged.
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
Vitorino, R., Krenkova, J., Foret, F., Domingues, P., Amado, F. (2011). Protein Identification Using Nano-HPLC-MS: ESI-MS and MALDI-MS Interfaces. In: Toms, S., Weil, R. (eds) Nanoproteomics. Methods in Molecular Biology, vol 790. Humana Press. https://doi.org/10.1007/978-1-61779-319-6_3
Download citation
DOI: https://doi.org/10.1007/978-1-61779-319-6_3
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-318-9
Online ISBN: 978-1-61779-319-6
eBook Packages: Springer Protocols