Proteomic research has proved valuable for understanding the molecular mechanisms of biological processes, as well as in the search for biomarkers for a variety of diseases which lack a molecular diagnostic. While several new approaches are being developed, two-dimensional (2-DE) gel electrophoresis is still one of the most commonly used techniques, despite its many limitations. However, for biomarker research, 2-DE gel electrophoresis alone does not fulfill the necessary pre-requisites. If such a technique is utilized exclusively, a great part of a given proteome remains unseen. Therefore, very precise and sensitive techniques are needed. Here, we present a brief review of known methodologies that try to overcome the limitations of conventional proteome analysis as well as their respective advantages and limitations. © The Author 2008. Published by Oxford University Press. All rights reserved.74312321Wilkins, M.R., Sanchez, J.C., Gooley, A.A., Progress with proteome projects: Why all proteins expressed by a genome should be identified and how to do it (1996) Biotechnol Genet Eng Rev, 13, pp. 19-50Klose, J., Nock, C., Herrmann, M., Genetic analysis of the mouse brain proteome (2002) Nat Genet, 30, pp. 385-393Lasonder, E., Ishihama, Y., Andersen, J.S., Analysis of the Plasmodium falciparum proteome by high-accuracy mass spectrometry (2002) Nature, 419, pp. 537-542Uetz, P., Giot, L., Cagney, G., A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae (2000) Nature, 403, pp. 623-627Ito, T., Chiba, T., Ozawa, R., A comprehensive two-hybrid analysis to explore the yeast protein interactome (2001) Proc Natl Acad Sci USA, 98, pp. 4569-4574Norin, M., Sundström, M., Structural proteomics: Developments in structure-to-function predictions (2002) Trends Biotechnol, 20, pp. 79-84Sali, A., Glaeser, R., Earnest, T., Baumeister, W., From words to literature in structural proteomics (2003) Nature, 422, pp. 216-225Gygi, S.P., Corthals, G.L., Zhang, Y., Evaluation of two-dimensional gel electrophoresis based proteome analysis tecnology (2000) Proc Natl Acad Sci USA, 97, pp. 9390-9395Washburn MP, Wolters D, Yates JR, 3rd. Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat Biotechnol 2001;19:242-7Wu, L., Han, D.K., Overcoming the dynamic range problem in mass spectrometry-based shotgun proteomics (2006) Expert Rev Proteomics, 3, pp. 611-619Futcher, B., Latter, G.I., Monardo, P., A sampling of the yeast proteome (1999) MCB, 19, pp. 7357-7376Badock, V., Steinhusen, U., Bommert, K., Prefractionation of protein samples for proteome analysis using reversed-phase high-performance liquid chromatography (2001) Electrophoresis, 22, pp. 2856-2864Bjellqvist, B., Ek, K., Righetti, P.G., Isoelectric focusing in immobilized pH gradients: Principle, methodology and some applications (1982) J Biochem Biophys Methods, 6, pp. 317-339Martins, D., Astua-Monge, G., Coletta-Filho, H.D., Absence of classical heat shock response in the citrus pathogen Xylella fastidiosa (2007) Curr Microbiol, 54, pp. 119-123Chen, F., Yuan, Y., Li, Q., Proteomic analysis of rice plasma membrane reveals proteins involved in early defense response to bacterial blight (2007) Proteomics, 7, pp. 1529-1539Qin, G., Tian, S., Chan, Z., Crucial role of antioxidant proteins and hydrolytic enzymes in pathogenicity of Penicillium expansum: Analysis based on proteomics approach (2007) Mol Cell Proteomics, 6, pp. 425-438Pei, H., Zhu, H., Zeng, S., Proteome analysis and tissue microarray for profiling protein markers associated with lymph node metastasis in colorectal cancer (2007) J Proteome Res, 6, pp. 2495-2501Sultana, R., Boyd-Kimball, D., Cai, J., Proteomics analysis of the Alzheimer's disease hippocampal proteome (2007) J Alzheimers Dis, 11, pp. 153-164De Maio, A., Heat shock proteins: Facts, thoughts, and dreams (1999) Shock, 11, pp. 1-12Righetti, P.G., Castagna, A., Herbert, B., How to bring the "unseen" proteome to the limelight via electrophoretic prefractionation techniques (2005) Biosci Rep, 25, pp. 3-17Righetti, P.G., Boschetti, E., Lomas, L., Protein Equalizer Technology: The quest for a "democratic proteome (2006) Proteomics, 6, pp. 3980-3992Galmarini, C.M., Galmarini, F.C., Multidrug resistance in cancer therapy: Role of the microenvironment (2003) Curr Opin Investig Drugs, 4, pp. 1416-1421Rabilloud, T., Solubilization of proteins for electrophoretic analyses (1996) Electrophoresis, 17, pp. 813-829Chevallet, M., Santoni, V., Poinas, A., New zwitterionic detergents improve the analysis of membrane proteins by two-dimensional electrophoresis (1998) Electrophoresis, 11, pp. 1901-1909Martins, D., Menezes de Oliveira, B., Dos Santos Farias, A., The use of ASB-14 in combination with CHAPS is the best for solubilization of human brain proteins for two-dimensional gel electrophoresis (2007) Brief Funct Genomic Proteomic, 6, pp. 70-75Wildgruber, R., Harder, A., Obermaier, C., Towards higher resolution: Two-dimensional electrophoresis of Saccharomyces cerevisiae proteins using overlapping narrow immobilized pH gradients (2000) Electrophoresis, 21, pp. 2610-2616Fey, S.J., Larsen, P.M., 2D or not 2D. Two-dimensional gel electrophoresis (2001) Curr Opin Chem Biol, 5, pp. 26-33Cordwell, S.J., Nouwens, A.S., Verrills, N.M., Subproteomics based upon protein cellular location and relative solubilities in conjunction with composite two-dimensional electrophoresis gels (2000) Electrophoresis, 21, pp. 1094-1103Westbrook, J.A., Wheeler, J.X., Wait, R., The human heart proteome: Two-dimensional maps using narrow-range immobilised pH gradients (2006) Electrophoresis, 27, pp. 1547-1555Westbrook, J.A., Yan, J.X., Wait, R., Zooming-in on the proteome: Very narrow-range immobilised pH gradients reveal more protein species and isoforms (2001) Electrophoresis, 22, pp. 2865-2871Corthals, G.L., Nelson, P.S., Large-scale proteomics and its future impact on medicine (2001) Pharmacogenomics J, 1, pp. 15-19Klose, J., Kobalz, U., Two-dimensional electrophoresis of proteins: An updated protocol and implications for a functional analysis of the genome (1995) Electrophoresis, 16, pp. 1034-1059O'Farrell, P.Z., Goodman, H.M., O'Farrell, P.H., High resolution two-dimensional electrophoresis of basic as well as acidic proteins (1977) Cell, 12, pp. 1133-1141Wittmann-Liebold, B., Graack, H.R., Pohl, T., Two-dimensional gel electrophoresis as tool for proteomics studies in combination with protein identification by mass spectrometry (2006) Proteomics, 6, pp. 4688-4703Chahed, K., Kabbage, M., Hamrita, B., Detection of protein alterations in male breast cancer using two dimensional gel electrophoresis and mass spectrometry: The involvement of several pathways in tumorigenesis (2008) Clin Chim Acta, 388, pp. 106-114Nowalk, A.J., Nolder, C., Clifton, D.R., Comparative proteome analysis of subcellular fractions from Borrelia burgdorferi by NEPHGE and IPG (2006) Proteomics, 6, pp. 2121-2134Jung, E., Heller, M., Sanchez, J.C., Proteomics meets cell biology: The establishment of subcellular proteomes (2000) Electrophoresis, 21, pp. 3369-3377Gerner, C., Sauermann, G., Nuclear matrix proteins specific for subtypes of human hematopoietic cells (1999) J Cell Biochem, 72, pp. 470-482Neubauer, G., King, A., Rappsilber, J., Mass spectrometry and EST-database searching allows characterization of the multi-protein spliceosome complex (1998) Nat Genet, 20, pp. 46-50Wigge, P.A., Jensen, O.N., Holmes, S., Analysis of the Saccharomyces spindle pole by matrix-assisted laser desorption/ ionization (MALDI) mass spectrometry (1998) J Cell Biol, 141, pp. 967-977Rout, M.P., Aitchison, J.D., Suprapto, A., The yeast nuclear pore complex: Composition, architecture, and transport mechanism (2000) J Cell Biol, 148, pp. 635-651Rigaut, G., Shevchenko, A., Rutz, B., A generic protein purification method for protein complex characterization and proteome exploration (1999) Nat Biotechnol, 17, pp. 1030-1032Havugimana, P.C., Wong, P., Emili, A., Improved proteomic discovery by sample pre-fractionation using dual-column ion-exchange high performance liquid chromatography (2007) J Chromatogr B Analyt Technol Biomed Life Sci, 847, pp. 54-61Görg, A., Boguth, G., Kopf, A., Sample prefractionation with Sephadex isoelectric focusing prior to narrow pH range two-dimensional gels (2002) Proteomics, 2, pp. 1652-1657Solassol, J., Marin, P., Demettre, E., Proteomic detection of prostate-specific antigen using a serum fractionation procedure: Potential implication for new low-abundance cancer biomarkers detection (2005) Anal Biochem, 338, pp. 26-31Martosella, J., Zolotarjova, N., Liu, H., High recovery HPLC separation of lipid rafts for membrane proteome analysis (2006) J Proteome Res, 5, pp. 1301-1312Echan, L.A., Tang, H.Y., Ali-Khan, N., Depletion of multiple high-abundance proteins improves protein profiling capacities of human serum and plasma (2005) Proteomics, 5, pp. 3292-3303Zolotarjova, N., Martosella, J., Nicol, G., Differences among techniques for high-abundant protein depletion (2005) Proteomics, 5, pp. 3304-3313Tanaka, Y., Akiyama, H., Kuroda, T., A novel approach and protocol for discovering extremely low-abundance proteins in serum (2006) Proteomics, 6, pp. 4845-4855Björhall, K., Miliotis, T., Davidsson, P., Comparison of different depletion strategies for improved resolution in proteomic analysis of human serum samples (2005) Proteomics, 5, pp. 307-317Granger, J., Siddiqui, J., Copeland, S., Albumin depletion of human plasma also removes low abundance proteins including the cytokines (2005) Proteomics, 5, pp. 4713-4718Veenstra, T.D., Conrads, T.P., Hood, B.L., Biomarkers: Mining the biofluid proteome (2005) Mol Cell Proteomics, 4, pp. 409-418Fountoulakis, M., Juranville, J.F., Jiang, L., Depletion of the high-abundance plasma proteins (2004) AminoAcids, 27, pp. 249-259Jmeian, Y., El Rassi, Z., Tandem affinity monolithic microcolumns with immobilized protein a, protein go,¢ and antibodies for depletion of high abundance proteins from serum samples: Integrated microcolumn-based fluidic system for simultaneous depletion and tryptic digestion (2007) J Proteome Res, 6, pp. 947-954Gong, Y., Li, X., Yang, B., Different immunoaffinity fractionation strategies to characterize the human plasma proteome (2006) J Proteome Res, 5, pp. 1379-1387Khan, A., Packer, N.H., Simple urinary sample preparation for proteomic analysis (2006) J Proteome Res, 5, pp. 2824-2838Steinberg, T.H., Jones, L.J., Haugland, R.P., SYPRO orange and SYPRO red protein gel stains: One-step flourescent staining of denaturing gels for detection of nanogram levels of protein (1996) Anal Biochem, 239, pp. 223-237Unlü, M., Morgan, M.E., Minden, J.S., Difference gel electrophoresis: A single gel method for detecting changes in protein extracts (1997) Electrophoresis, 18, pp. 2071-2077Chan, H.L., Gharbi, S., Gaffney, P.R., Proteomic analysis of redox- and ErbB2-dependent changes in mammary luminal epithelial cells using cysteine- and lysine-labelling two-dimensional difference gel electrophoresis (2005) Proteomics, 5, pp. 2908-2926Alban, A., David, S.O., Bjorkesten, L., A novel experimental design for comparative two-dimensional gel analysis: Two-dimensional difference gel electrophoresis incorporating a pooled internal standard (2003) Proteomics, 3, pp. 36-44Marouga, R., David, S., Hawkins, E., The development of the DIGE system: 2D fluorescence difference gel analysis technology (2005) Anal Bioanal Chem, 382, pp. 669-678Schüpbach, J., Ammann, R.W., Freiburghaus, A.U., A universal method for two-dimensional polyacrylamide gel electrophoresis of membrane proteins using isoelectric focusing on immobilized pH gradients in the first dimension (1991) Anal Biochem, 196, pp. 337-343Molloy, M.P., Herbert, B.R., Walsh, B.J., Extraction of membrane proteins by differential solubilization for separation using two-dimensional gel electrophoresis (1998) Electrophoresis, 19, pp. 837-844Deshusses, J.M., Burgess, J.A., Scherl, A., Exploitation of specific properties of trifluoroethanol for extraction and separation of membrane proteins (2003) Proteomics, 3, pp. 1418-1424Molloy, M.P., Herbert, B.R., Slade, M.B., Proteomic analysis of the Escherichia coh outer membrane (2000) Eur J Biochem, 267, pp. 2871-2881Gazzana, G., Borlak, J., Improved method for proteome mapping of the liver by 2-DE MALDI-TOF MS (2007) J Proteome Res, 6, pp. 3143-3151Link, A.J., Eng, J., Schieltz, D.M., Direct analysis of protein complexes using mass spectrometry (1999) Nat Biotechnol, 17, pp. 676-682Link, A.J., Multidimensional peptide separations in proteomics (2002) Trends Biotechnol, 20 (SUPPL. 12), pp. S8-13Haas, W., Faherty, B.K., Gerber, S.A., Optimization and use of peptide mass measurement accuracy in shotgun proteomics (2006) Mol Cell Proteomics, 5, pp. 1326-1337Nesvizhskii, A.I., Protein identification by tandem mass spectrometry and sequence database searching (2007) Methods Mol Biol, 367, pp. 87-119Gygi, S.P., Rist, B., Gerber, S.A., Quantitative analysis of complex protein mixtures using isotope-coded affinity tags (1999) Nat Biotechnol, 17, pp. 994-999Goodlett, D.R., Keller, A., Watts, J.D., Differential stable isotope labeling of peptides for quantitation and de novo sequence derivation (2001) Rapid Commun Mass Spectrom, 15, pp. 1214-1221Ross, P.L., Huang, Y.N., Marchese, J.N., Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents (2004) Mol Cell Proteomics, 3, pp. 1154-1169Schmidt, A., Kellermann, J., Lottspeich, F., A novel strategy for quantitative proteomics using isotope-coded protein labels (2005) Proteomics, 5, pp. 4-15Bledi, Y., Inberg, A., Linial, M., PROCEED: A proteomic method for analysing plasma membrane proteins in living mammalian cells (2003) Briefings Funct Genomics Proteomics, 2, pp. 254-265Chen, E.I., Hewel, J., Felding-Habermann, B., Large scale protein profiling by combination of protein fractionation and multidimensional protein identification technology (MudPIT) (2006) Mol Cell Proteomics, 5, pp. 53-56Nunn, B.L., Shaffer, S.A., Scherl, A., Comparison of a Salmonella typhimurium proteome defined by shotgun proteomics directly on an LTQ-FT and by proteome pre-fractionation on an LCQ-DUO (2006) Brief Funct Genomic Proteomic, 5, pp. 154-168Li, S., Wang, J., Zhang, X., Proteomic characterization of two snake venoms: Naja naja atra and Agkistrodon halys (2004) Biochem J, 384, pp. 119-127Kubota, K., Kosaka, T., Ichikawa, K., Combination of two-dimensional electrophoresis and shotgun peptide sequencing in comparative proteomics (2005) J Chromatogr B Analyt Technol Biomed Life Sci, 815, pp. 3-9Wingren, C., Borrebaeck, C.A., High-throughput proteomics using antibody microarrays (2004) Expert Rev Proteomics, 1, pp. 355-364Dexlin, L., Ingvarsson, J., Frendéus, B., Design of recombinant antibody microarrays for cell surface membrane proteomics (2008) J Proteome Res, 7, pp. 319-327Davies, D.H., Wyatt, L.S., Newman, F.K., Antibody profiling by proteome microarray reveals the immunogenicity of the attenuated smallpox vaccine, modified vaccinia virus ankara (MVA) is comparable to dryvax(R) (2008) J Virol, 82, pp. 652-663Sze, S.K., de Kleijn, D.P., Lai, R.C., Elucidating the secretion proteome of human embryonic stem cell-derived mesenchymal stem cells (2007) Mol Cell Proteomics, 6, pp. 1680-1689Wingren, C., Ingvarsson, J., Dexlin, L., Design of recombinant antibody microarrays for complex proteome analysis: Choice of sample labeling-tag and solid support (2007) Proteomics, 7, pp. 3055-3065