Abstract
This chapter describes the development of the two-dimensional separation methods using flow field-flow fractionation (F4) and isoelectric focusing for proteomics utility. The methods described here are the rapid, non-gel-based, on-line, two-dimensional separation methods in which proteins are separated by isoelectric focusing (IEF) in the first dimension according to differences in isoelectric point (pI) followed by size based separation using F4 (either hollow fiber F4 or multilane asymmetrical F4 channels) in an orthogonal direction. In this chapter, the capillary IEF-HF5 and IEF-AF4 methods are described with the demonstration of system performances using protein standards. Also described are the applications to human urinary proteome samples in which proteome fractions are collected and tryptic digested for the proteomic analysis using nanoflow liquid chromatography-tandem mass spectrometry (nLC-ESI-MS-MS).
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References
Giddings JC (1993) Field-flow fractionation: analysis of macromolecular, colloidal, and particulate materials. Science 260:1456–1465
Ratanathanawongs-Williams SK (2000) Flow field-flow fractionation. In: Schimpf ME, Caldwell KD, Giddings JC (eds) Field-flow fractionation handbook. Wiley-Interscience, New York
Wahlund KG, Giddings JC (1987) Properties of an asymmetrical flow field-flow fractionation channel having one permeable wall. Anal Chem 59:1332–1339
Moon MH, Kwon HS, Park I (1997) Stopless flow injection in asymmetrical flow field-flow fractionation using a frit inlet. Anal Chem 69:1436–1440
Jönsson JA, Carlshaf A (1989) Flow field-flow fractionation in hollow cylindrical fibers. Anal Chem 61:11–18
Lee WJ, Min BR, Moon MH (1999) Improvement in particle separation by hollow fiber flow field-flow fractionation and the potential use in obtaining particle size distribution. Anal Chem 71:3446–3452
Bruijnsvoort MV, Kok WT, Tijssen R (2001) Hollow-fiber flow field-flow fractionation of synthetic polymers in organic solvents. Anal Chem 73:4736–4742
Kang D, Oh S, Reschiglian P, Moon MH (2008) Separation of mitochondria by flow field-flow fractionation for proteomic analysis. Analyst 133:505–515
Oh S, Kang D, Ahn SM, Simpson RJ, Lee BH, Moon MH (2007) Miniaturized asymmetrical flow field-flow fractionation: application to biological vesicles. J Sep Sci 30:1082–1087
Kang D, Oh S, Ahn SM, Lee BH, Moon MH (2008) Proteomic analysis of exosomes from human neural stem cells by flow field-flow fractionation and nanoflow liquid chromatography − tandem mass spectrometry. J Proteome Res 7:3475–3480
Kang D, Yoo JS, Kim MO, Moon MH (2009) A soft preparative method for membrane proteome analysis using frit inlet asymmetrical flow field-flow fractionation: application in a prostatic cancer cell line. J Proteome Res 8:982–991
Kang D, Ji E, Moon MH, Yoo JS (2010) Lectin based enrichment method for glycoproteomics using hollow fiber flow field-flow fractionation: application to streptococcus pyogenes. J Proteome Res. (in Press)
Righetti PG, Castagna A, Herbert B (2001) Peer reviewed: prefractionation techniques in proteome analysis. A new approach identifies more low-abundance proteins. Anal Chem 73:320A–326A
Zhou F, Johnston MV (2004) Protein characterization by on-line capillary isoelectric focusing, reversed-phase liquid chromatography, and mass spectrometry. Anal Chem 76:2734–2740
Klose J, Kobalz U (1995) Two-dimensional electrophoresis of proteins: an updated protocol and implications for a functional analysis of the genome. Electrophoresis 16:1034–1059
Giddings JC (1991) Unified separation science. John, New York, pp 126–128
Chen J, Lee CS, Shen Y, Smith RD, Baehrecke EH (2002) Integration of capillary isoelectric focusing with capillary reversed-phase liquid chromatography for two-dimensional proteomics separation. Electrophoresis 23:3143–3148
Chen J, Balgley BM, DeVoe DL, Lee CS (2003) Capillary isoelectric focusing-based multidimensional concentration/separation platform for proteome analysis. Anal Chem 75:3145–3152
Tragas C, Pawliszyn J (2000) On-line coupling of high performance gel filtration chromatography with imaged capillary isoelectric focusing using a membrane interface. Electrophoresis 21:227–237
Yang C, Zhang L, Liu H, Zhang W, Zhang Y (2003) Two-dimensional capillary electrophoresis involving capillary isoelectric focusing and capillary zone electrophoresis. J Chromatogr A 1018:97–103
Yang C, Liu H, Yang Q, Zhang L, Zhang W, Zhang Y (2003) On-line hyphenation of capillary isoelectric focusing and capillary gel electrophoresis by a dialysis interface. Anal Chem 75:215–218
Kang D, Moon MH (2006) Development of non-gel-based two-dimensional separation of intact proteins by an on-line hyphenation of capillary isoelectric focusing and hollow fiber flow field-flow fractionation. Anal Chem 78:5789–5798
Kim KH, Moon MH (2009) Development of a multilane channel system for nongel-based two-dimensional protein separations using isoelectric focusing and asymmetrical flow field-flow fractionation. Anal Chem 81:1715–1721
Kim KH, Moon MH (2009) High speed two-dimensional protein separation without gel by isoelectric focusing-asymmetrical flow field-flow fractionation:application to urinary proteome. J Proteome Res 8:4272–4278
Reschiglian P, Zattoni A, Cinque L, Roda B, Piaz FD, Roda A, Moon MH, Min BR (2004) Hollow-fiber flow field-flow fractionation for whole bacteria analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Anal Chem 76:2103–2111
Pang JX, Gianni N, Dongre AR, Hefta SA, Opiteck GJ (2002) Biomarker discovery in urine by proteomics. J Proteome Res 1:161–169
Kang D, Nam H, Kim YS, Moon MH (2005) Dual-purpose sample trap for on-line strong cation-exchange chromatography/reversed-phase liquid chromatography/tandem mass spectrometry for shotgun proteomics application to the human jurkat T-cell proteome. J Chromatogr A 1070:193–200
Castagna A, Cecconi D, Sennels L, Rappsilber J, Guerrier L, Fortis F, Boschetti E, Lomas L, Righetti PG (2005) Exploring the hidden human urinary proteome via ligand library beads. J Proteome Res 4:1917–1930
Coon JJ, Zürbig P, Dakna M, Dominiczak AF, Decramer S, Fliser D, Frommberger M, Golovko I, Good DM, Herget-Rosenthal S, Jankowski J, Julian BA, Kellmann M, Kolch W, Massy Z, Novak J, Rossing K, Schanstra JP, Schiffer E, Theodorescu D, Vanholder R, Weissinger EM, Mischak H, Schmitt-Kopplin P (2008) CE-MS analysis of the human urinary proteome for biomarker discovery and disease diagnostics. Proteomics 2:964–973
Spahr CS, Davis MT, McGinley MD, Robinson JH, Bures EJ, Beierle J, Mort J, Courchesne PL, Chen K, Wahl RC, Yu W, Luethy R, Patterson SD (2001) Towards defining the urinary proteome using liquid chromatography-tandem mass spectrometry. I. Profiling an unfractionated tryptic digest. Proteomics 1:93–107
Pieper R, Gatlin CL, McGrath AM, Makusky AJ, Mondal M, Seonarain M, Field E, Schatz CR, Estock MA, Ahmed N, Anderson NG, Steiner S (2004) Characterization of the human urinary proteome: a method for high-resolution display of urinary proteins on two-dimensional electrophoresis gels with a yield of nearly 1400 distinct protein spots. Proteomics 4:1159–1174
Adachi J, Kumar C, Zhang Y, Olsen JV, Mann M (2006) The human urinary proteome contains more than 1500 proteins, including a large proportion of membrane proteins. Genome Biol 7:R80
Arita K, South AP, Hans-Filho G, Sakuma TH, Lai-Cheong J, Clements S, Odashiro M, Odashiro DN, Hans-Neto G, Hans NR, Holder MV, Bhogal BS, Hartshorne ST, Akiyama M, Shimizu H, McGrath JA (2008) Oncostatin M receptor-beta mutations underlie familial primary localized cutaneous amyloidosis. Am J Hum Genet 82:73–80
Lai LW, Whitehair O, Wu MJ, O’Meara M, Lien YH (2003) Analysis of splice-site mutations of the alpha-galactosidase A gene in Fabry disease. Clin Genet 63:476–482
Steinfeld R, Reinhardt K, Schreiber K, Hillebrand M, Kraetzner R, Bruck W, Saftig P, Gartner J (2006) Cathepsin D deficiency is associated with a human neurodegenerative disorder. Am J Hum Genet 78:988–998
Aminoff M, Carter JE, Chadwick RB, Johnson C, Grasbeck R, Abdelaal MA, Broch H, Jenner LB, Verroust PJ, Moestrup SK, de la Chapelle A, Krahe R (1999) Mutations in CUBN, encoding the intrinsic factor-vitamin B12 receptor, cubilin, cause hereditary megaloblastic anaemia 1. Nat Genet 21:309–313
Acknowledgments
This work was supported by a Korea National Research Foundation Grant (NRF-2010-0014046).
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Moon, M.H., Kim, K.H., Kang, D. (2012). Two-Dimensional Separation for Proteomic Analysis. In: Williams, S., Caldwell, K. (eds) Field-Flow Fractionation in Biopolymer Analysis. Springer, Vienna. https://doi.org/10.1007/978-3-7091-0154-4_4
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DOI: https://doi.org/10.1007/978-3-7091-0154-4_4
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