Protein and peptide fractionation, enrichment and depletion: Tools for the complex proteome

The identification, quantitation and global characterisation of all proteins within a given proteome are extremely challenging. This is due to the absolute detection limits of technology as well as the dynamic range in expression of proteins; and the extreme diversity and heterogeneity of the proteome. To overcome such issues, the use of separation technologies has played a critical role in reducing sample complexity. To date, a plethora of chromatographic and electrophoretic fractionation tools have evolved over the years assisting in simplifying complex protein and peptide mixtures. Here, we review a range of these technologies highlighting the challenges of protein and peptide analysis in the context of proteome research and some of the advantages and disadvantages of present techniques.

[1]  M. Mann,et al.  Proteomic analysis of post-translational modifications , 2003, Nature Biotechnology.

[2]  D. Hochstrasser,et al.  From Proteins to Proteomes: Large Scale Protein Identification by Two-Dimensional Electrophoresis and Arnino Acid Analysis , 1996, Bio/Technology.

[3]  P. Righetti,et al.  Sherlock Holmes and the proteome − a detective story , 2007, The FEBS journal.

[4]  Chen Li,et al.  Comparative proteomic study of two closely related ovarian endometrioid adenocarcinoma cell lines using cIEF fractionation and pathway analysis , 2009, Electrophoresis.

[5]  Qing‐Yu He,et al.  Heparin chromatography to deplete high-abundance proteins for serum proteomics. , 2008, Clinica chimica acta; international journal of clinical chemistry.

[6]  K. Mechtler,et al.  Enhanced detection and identification of multiply phosphorylated peptides using TiO2 enrichment in combination with MALDI TOF/TOF MS , 2008, Proteomics.

[7]  J. Shabanowitz,et al.  Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae , 2002, Nature Biotechnology.

[8]  Nicholas A. Cellar,et al.  Immunodepletion of high abundance proteins coupled on-line with reversed-phase liquid chromatography: a two-dimensional LC sample enrichment and fractionation technique for mammalian proteomics. , 2009, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[9]  R. Lindmark,et al.  Binding of immunoglobulins to protein A and immunoglobulin levels in mammalian sera. , 1983, Journal of immunological methods.

[10]  Eugene A. Kapp,et al.  Overview of the HUPO Plasma Proteome Project: Results from the pilot phase with 35 collaborating laboratories and multiple analytical groups, generating a core dataset of 3020 proteins and a publicly‐available database , 2005, Proteomics.

[11]  R. Vera-Estrella,et al.  Quantitative Proteomics of the Tonoplast Reveals a Role for Glycolytic Enzymes in Salt Tolerance[C][W] , 2009, The Plant Cell Online.

[12]  P. Righetti,et al.  Isoelectric beads for proteome pre‐fractionation. II: Experimental evaluation in a multicompartment electrolyzer , 2005, Proteomics.

[13]  Robert Tonge,et al.  Evaluation of saturation labelling two‐dimensional difference gel electrophoresis fluorescent dyes , 2003, Proteomics.

[14]  Richard D. Smith,et al.  Ultrahigh-throughput proteomics using fast RPLC separations with ESI-MS/MS. , 2005, Analytical chemistry.

[15]  P. Righetti,et al.  A turning point in proteome analysis: Sample prefractionation via multicompartment electrolyzers with isoelectric membranes , 2000, Electrophoresis.

[16]  M. Larsen,et al.  Highly selective enrichment of phosphorylated peptides using titanium dioxide , 2006, Nature Protocols.

[17]  H. Gross,et al.  Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels , 1987 .

[18]  E. Nägele,et al.  Two-dimensional nano-liquid chromatography-mass spectrometry system for applications in proteomics. , 2003, Journal of chromatography. A.

[19]  Yong-Ak Song,et al.  Free-flow zone electrophoresis of peptides and proteins in PDMS microchip for narrow pI range sample prefractionation coupled with mass spectrometry. , 2010, Analytical chemistry.

[20]  C. Eckerskorn,et al.  Peroxisomes from the heavy mitochondrial fraction: isolation by zonal free flow electrophoresis and quantitative mass spectrometrical characterization. , 2010, Journal of proteome research.

[21]  Eberhard Durr,et al.  Direct proteomic mapping of the lung microvascular endothelial cell surface in vivo and in cell culture , 2004, Nature Biotechnology.

[22]  Wei-Wei Zhang,et al.  Affinity separation: divide and conquer the proteome. , 2004, Drug discovery today. Technologies.

[23]  Matthew Davison,et al.  Validation and development of fluorescence two‐dimensional differential gel electrophoresis proteomics technology , 2001, Proteomics.

[24]  Jie Zhang,et al.  Protein identification assisted by the prediction of retention time in liquid chromatography/tandem mass spectrometry. , 2005, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[25]  P. Righetti,et al.  The ProteoMiner in the proteomic arena: a non-depleting tool for discovering low-abundance species. , 2008, Journal of proteomics.

[26]  Yu‐Ju Chen,et al.  Identification of in vivo phosphorylation sites of lens proteins from porcine eye lenses by a gel-free phosphoproteomics approach , 2010, Molecular vision.

[27]  E. Harrison,et al.  Rat plasma proteomics: effects of abundant protein depletion on proteomic analysis. , 2007, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[28]  R. Wildgruber,et al.  Free-flow electrophoresis system for proteomics applications. , 2008, Methods in molecular biology.

[29]  M. Larsen,et al.  Characterization of the human cerebrospinal fluid phosphoproteome by titanium dioxide affinity chromatography and mass spectrometry. , 2008, Analytical chemistry.

[30]  P. Malfertheiner,et al.  Identification and confirmation of increased fibrinopeptide a serum protein levels in gastric cancer sera by magnet bead assisted MALDI-TOF mass spectrometry. , 2006, Journal of proteome research.

[31]  S. Gygi,et al.  Proteomics: the move to mixtures. , 2001, Journal of mass spectrometry : JMS.

[32]  Martin R Larsen,et al.  TiO(2)-based phosphoproteomic analysis of the plasma membrane and the effects of phosphatase inhibitor treatment. , 2008, Journal of proteome research.

[33]  D. Hochstrasser,et al.  The dynamic range of protein expression: A challenge for proteomic research , 2000, Electrophoresis.

[34]  Hildegard Geyer,et al.  Strategies for analysis of glycoprotein glycosylation. , 2006, Biochimica et biophysica acta.

[35]  Yan Gao,et al.  HUPO BPP pilot study: A proteomics analysis of the mouse brain of different developmental stages , 2007, Proteomics.

[36]  Egisto Boschetti,et al.  Prefractionation techniques in proteome analysis: The mining tools of the third millennium , 2005, Electrophoresis.

[37]  Madalina M. Drugan,et al.  Strong Cation Exchange-based Fractionation of Lys-N-generated Peptides Facilitates the Targeted Analysis of Post-translational Modifications* , 2009, Molecular & Cellular Proteomics.

[38]  S. Terabe,et al.  Sample enrichment techniques in capillary electrophoresis: focus on peptides and proteins. , 2006, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[39]  A. Guttman,et al.  A fully automated 2‐D LC‐MS method utilizing online continuous pH and RP gradients for global proteome analysis , 2007, Electrophoresis.

[40]  S. Kern,et al.  Quality control of serum albumin depletion for proteomic analysis. , 2007, Clinical chemistry.

[41]  H. Mottaz,et al.  Using size exclusion chromatography‐RPLC and RPLC‐CIEF as two‐dimensional separation strategies for protein profiling , 2006, Electrophoresis.

[42]  Pengyuan Yang,et al.  Large scale depletion of the high‐abundance proteins and analysis of middle‐ and low‐abundance proteins in human liver proteome by multidimensional liquid chromatography , 2008, Proteomics.

[43]  K. Blennow,et al.  Evaluation of sample fractionation using micro-scale liquid-phase isoelectric focusing on mass spectrometric identification and quantitation of proteins in a SILAC experiment. , 2007, Rapid communications in mass spectrometry : RCM.

[44]  Marcel Ottens,et al.  pH-gradient ion-exchange chromatography: an analytical tool for design and optimization of protein separations. , 2007, Journal of chromatography. A.

[45]  G. Lajoie,et al.  An integrated procedure of selective injection, sample stacking and fractionation of phosphopeptides for MALDI MS analysis. , 2007, Analytica chimica acta.

[46]  Rong Zeng,et al.  Human plasma proteome analysis by multidimensional chromatography prefractionation and linear ion trap mass spectrometry identification. , 2005, Journal of proteome research.

[47]  S. Terabe,et al.  Field-enhanced sample injection for high-sensitivity analysis of peptides and proteins in capillary electrophoresis-mass spectrometry. , 2004, Journal of chromatography. A.

[48]  F. Zhou,et al.  Intact protein profiling of Chlorobium tepidum by capillary isoelectric focusing, reversed-phase liquid chromatography, and mass spectrometry. , 2007, Analytical Chemistry.

[49]  S. Lemeer,et al.  Online automated in vivo zebrafish phosphoproteomics: from large-scale analysis down to a single embryo. , 2008, Journal of proteome research.

[50]  P. Schmitt‐Kopplin,et al.  Capillary electrophoresis – mass spectrometry: 15 years of developments and applications , 2003, Electrophoresis.

[51]  G. Karlsson,et al.  Separation of latent, prelatent, and native forms of human antithrombin by heparin affinity high-performance liquid chromatography. , 2004, Protein expression and purification.

[52]  D. Figeys,et al.  Multiplexed proteomic reactor for the processing of proteomic samples. , 2007, Analytical chemistry.

[53]  Kevin S. Shores,et al.  Shotgun proteomic analysis of cerebrospinal fluid using off-gel electrophoresis as the first-dimension separation. , 2008, Journal of proteome research.

[54]  John P Cortens,et al.  Use of peptide retention time prediction for protein identification by off-line reversed-phase HPLC-MALDI MS/MS. , 2006, Analytical chemistry.

[55]  D. Kaniansky,et al.  Fractionation of glycoforms of recombinant human erythropoietin by preparative capillary isotachophoresis , 2005, Electrophoresis.

[56]  D. DeVoe,et al.  Membrane proteome analysis of microdissected ovarian tumor tissues using capillary isoelectric focusing/reversed-phase liquid chromatography-tandem MS. , 2007, Analytical chemistry.

[57]  Helmut E Meyer,et al.  Multidimensional nano-HPLC for analysis of protein complexes , 2003, Journal of the American Society for Mass Spectrometry.

[58]  A. Heck,et al.  Selective isolation at the femtomole level of phosphopeptides from proteolytic digests using 2D-NanoLC-ESI-MS/MS and titanium oxide precolumns. , 2004, Analytical chemistry.

[59]  D. Herold,et al.  Immunoaffinity separation of plasma proteins by IgY microbeads: Meeting the needs of proteomic sample preparation and analysis , 2005, Proteomics.

[60]  J. Carmody,et al.  Mass profiling‐directed isolation and identification of a stage‐specific serologic protein biomarker of advanced prostate cancer , 2005, Proteomics.

[61]  Yu-Fen Huang,et al.  Capillary electrophoresis‐based separation techniques for the analysis of proteins , 2006, Electrophoresis.

[62]  P. Righetti,et al.  A pI-based protein fractionation method using solid-state buffers. , 2008, Journal of proteomics.

[63]  K. Mechtler,et al.  Automated, on‐line two‐dimensional nano liquid chromatography tandem mass spectrometry for rapid analysis of complex protein digests , 2004, Proteomics.

[64]  K. Campbell,et al.  Proteomic analysis of plasma membrane and secretory vesicles from human neutrophils , 2007, Proteome Science.

[65]  V. Wasinger,et al.  Peptide enrichment and protein fractionation using selective electrophoresis , 2008, Proteomics.

[66]  I. Arnaud,et al.  Protein fractionation in a multicompartment device using Off‐Gel™ isoelectric focusing , 2003, Electrophoresis.

[67]  G. Lubec,et al.  Changes in the levels of low-abundance brain proteins induced by kainic acid. , 2001, European journal of biochemistry.

[68]  J. Myung,et al.  Use of solution-IEF-fractionation leads to separation of 2673 mouse brain proteins including 255 hydrophobic structures. , 2006, Journal of proteome research.

[69]  Tyge Greibrokk,et al.  Two-dimensional capillary liquid chromatography: pH gradient ion exchange and reversed phase chromatography for rapid separation of proteins. , 2006, Journal of chromatography. A.

[70]  Thomas P Conrads,et al.  Methods for fractionation, separation and profiling of proteins and peptides , 2002, Electrophoresis.

[71]  Veronika A. Glukhova,et al.  Contribution of protein fractionation to depth of analysis of the serum and plasma proteomes. , 2007, Journal of proteome research.

[72]  Richard D. Smith,et al.  Combining capillary electrophoresis with mass spectrometry for applications in proteomics , 2005, Electrophoresis.

[73]  K. Jensen,et al.  Microfluidic preparative free-flow isoelectric focusing: system optimization for protein complex separation. , 2010, Analytical chemistry.

[74]  Dietrich Kohlheyer,et al.  Microfluidic high-resolution free-flow isoelectric focusing. , 2007, Analytical chemistry.

[75]  Klavs F Jensen,et al.  Cascaded free-flow isoelectric focusing for improved focusing speed and resolution. , 2007, Analytical chemistry.

[76]  J. Jorgenson,et al.  Comprehensive three-dimensional separation of peptides using size exclusion chromatography/reversed phase liquid chromatography/optically gated capillary zone electrophoresis. , 1995, Analytical chemistry.

[77]  Dario Neri,et al.  In vivo protein biotinylation for identification of organ-specific antigens accessible from the vasculature , 2005, Nature Methods.

[78]  D. Lubman,et al.  Micro-proteome analysis using micro-chromatofocusing in intact protein separations. , 2008, Journal of chromatography. A.

[79]  D. Speicher,et al.  Enhanced analysis of human breast cancer proteomes using micro-scale solution isoelectrofocusing combined with high resolution 1-D and 2-D gels. , 2002, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[80]  N. Anderson,et al.  The Human Plasma Proteome: History, Character, and Diagnostic Prospects , 2003, Molecular & Cellular Proteomics.

[81]  D. Speicher,et al.  A novel four‐dimensional strategy combining protein and peptide separation methods enables detection of low‐abundance proteins in human plasma and serum proteomes , 2005, Proteomics.

[82]  M. Mann,et al.  4. Proteomic Analysis of Posttranslational Modifications , 2013 .

[83]  A. Staby,et al.  Comparison of chromatographic ion-exchange resins IV. Strong and weak cation-exchange resins and heparin resins. , 2005, Journal of chromatography. A.

[84]  A. A. Farooqui Purification of enzymes by heparin-sepharose affinity chromatography. , 1980, Journal of chromatography.

[85]  Pier Giorgio Righetti,et al.  Prefractionation techniques in proteome analysis , 2001, Proteomics.

[86]  D. Desiderio,et al.  Proteomics analysis of prefractionated human lumbar cerebrospinal fluid , 2005, Proteomics.

[87]  Yu-Chang Tyan,et al.  Proteomic profiling of human urinary proteome using nano-high performance liquid chromatography/electrospray ionization tandem mass spectrometry. , 2006, Analytica chimica acta.

[88]  John R Yates,et al.  Combining protein-based IMAC, peptide-based IMAC, and MudPIT for efficient phosphoproteomic analysis. , 2008, Journal of proteome research.

[89]  Albert Sickmann,et al.  Enhanced N-Glycosylation Site Analysis of Sialoglycopeptides by Strong Cation Exchange Prefractionation Applied to Platelet Plasma Membranes *S , 2007, Molecular & Cellular Proteomics.

[90]  A. Görg,et al.  Current two‐dimensional electrophoresis technology for proteomics , 2004, Proteomics.

[91]  Juri Rappsilber,et al.  Proteomic analysis of human blood serum using peptide library beads. , 2007, Journal of proteome research.

[92]  M. Mann,et al.  Peptide separation with immobilized pI strips is an attractive alternative to in‐gel protein digestion for proteome analysis , 2008, Proteomics.

[93]  M. Bier,et al.  Recycling isoelectric focusing and isotachophoresis , 1989 .

[94]  A. Bacic,et al.  Analysis of the Oryza sativa plasma membrane proteome using combined protein and peptide fractionation approaches in conjunction with mass spectrometry. , 2008, Journal of proteome research.

[95]  Ronald J Moore,et al.  Multidimensional proteome analysis of human mammary epithelial cells. , 2004, Journal of proteome research.

[96]  C. Gelfand,et al.  Application of free flow electrophoresis to the analysis of the urine proteome. , 2010, Methods in molecular biology.

[97]  Fuchu He,et al.  Different immunoaffinity fractionation strategies to characterize the human plasma proteome. , 2006, Journal of proteome research.

[98]  S. Lemeer,et al.  A versatile peptide pI calculator for phosphorylated and N‐terminal acetylated peptides experimentally tested using peptide isoelectric focusing , 2008, Proteomics.

[99]  M. Wilkins,et al.  Progress with gene‐product mapping of the Mollicutes: Mycoplasma genitalium , 1995, Electrophoresis.

[100]  P. Righetti,et al.  pI-based fractionation of serum proteomes versus anion exchange after enhancement of low-abundance proteins by means of peptide libraries. , 2009, Journal of proteomics.

[101]  M. Trivella,et al.  A gel-free approach in vascular smooth muscle cell proteome: perspectives for a better insight into activation , 2010, Proteome Science.

[102]  C. Hocart,et al.  The Medicago truncatula small protein proteome and peptidome. , 2006, Journal of proteome research.

[103]  Ronald J Moore,et al.  Ultra-high-efficiency strong cation exchange LC/RPLC/MS/MS for high dynamic range characterization of the human plasma proteome. , 2004, Analytical chemistry.

[104]  Free flow electrophoresis coupled with liquid chromatography-mass spectrometry for a proteomic study of the human cell line (K562/CR3). , 2004, Journal of chromatography. A.

[105]  G. Nicol,et al.  Reversed-phase high-performance liquid chromatographic prefractionation of immunodepleted human serum proteins to enhance mass spectrometry identification of lower-abundant proteins. , 2005, Journal of proteome research.

[106]  C. Giometti,et al.  A combinatorial approach to studying protein complex composition by employing size-exclusion chromatography and proteome analysis. , 2007, Journal of separation science.

[107]  S. Watson,et al.  A Comprehensive Proteomics and Genomics Analysis Reveals Novel Transmembrane Proteins in Human Platelets and Mouse Megakaryocytes Including G6b-B, a Novel Immunoreceptor Tyrosine-based Inhibitory Motif Protein*S , 2007, Molecular & Cellular Proteomics.

[108]  Rong Zeng,et al.  Fractionation of complex protein mixture by virtual three-dimensional liquid chromatography based on combined pH and salt steps. , 2008, Journal of proteome research.

[109]  P. Roepstorff,et al.  Highly Selective Enrichment of Phosphorylated Peptides from Peptide Mixtures Using Titanium Dioxide Microcolumns* , 2005, Molecular & Cellular Proteomics.

[110]  J. Carlis,et al.  Proteomics Analysis of Cells in Whole Saliva from Oral Cancer Patients via Value-added Three-dimensional Peptide Fractionation and Tandem Mass Spectrometry*S , 2008, Molecular & Cellular Proteomics.

[111]  Li Zang,et al.  A miniaturized multichamber solution isoelectric focusing device for separation of protein digests , 2002, Electrophoresis.

[112]  Anna M. Fitzgerald,et al.  Prefractionation, enrichment, desalting and depleting of low volume and low abundance proteins and peptides using the MF10. , 2008, Methods in molecular biology.

[113]  David J. Anderson,et al.  Gradient chromatofocusing. versatile pH gradient separation of proteins in ion-exchange HPLC: characterization studies. , 2002, Analytical chemistry.

[114]  Peter Karuso,et al.  A fluorescent natural product for ultra sensitive detection of proteins in one‐dimensional and two‐dimensional gel electrophoresis , 2003, Proteomics.

[115]  Dennis van Hoof,et al.  Concise Review: Trends in Stem Cell Proteomics , 2007, Stem cells.

[116]  J. Brand,et al.  Depletion efficiency and recovery of trace markers from a multiparameter immunodepletion column , 2006, Proteomics.

[117]  A. Namane,et al.  Phosphoproteomic analysis of Leishmania donovani pro‐ and amastigote stages , 2008, Proteomics.

[118]  Y. Rikihisa,et al.  Identification of Novel Surface Proteins of Anaplasma phagocytophilum by Affinity Purification and Proteomics , 2007, Journal of bacteriology.

[119]  Richard D. Smith,et al.  Integration of capillary isoelectric focusing with capillary reversed‐phase liquid chromatography for two‐dimensional proteomics separation , 2002, Electrophoresis.

[120]  T. Veenstra,et al.  Two‐dimensional liquid chromatography‐capillary zone electrophoresis‐sheathless electrospray ionization‐mass spectrometry: Evaluation for peptide analysis and protein identification , 2004, Electrophoresis.

[121]  E. De Pauw,et al.  Cell membrane proteomic analysis identifies proteins differentially expressed in osteotropic human breast cancer cells. , 2008, Neoplasia.

[122]  M. Ouellette,et al.  A combined proteomic and transcriptomic approach to the study of stage differentiation in Leishmania infantum , 2006, Proteomics.

[123]  Sunny Tam Electrophoretic prefractionation: new commercial tools from an old concept , 2006, Expert review of proteomics.

[124]  Wayne F. Patton,et al.  Detection technologies in proteome analysis. , 2002, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[125]  P. Schoenmakers,et al.  Optimizing the peak capacity per unit time in one-dimensional and off-line two-dimensional liquid chromatography for the separation of complex peptide samples. , 2009, Journal of chromatography. A.

[126]  L. Björck,et al.  Purification and some properties of streptococcal protein G, a novel IgG-binding reagent. , 1984, Journal of immunology.

[127]  V. Neuhoff,et al.  Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G‐250 and R‐250 , 1988, Electrophoresis.

[128]  L. Zolla,et al.  Capturing and amplifying impurities from purified recombinant monoclonal antibodies via peptide library beads: A proteomic study , 2007, Proteomics.

[129]  R. Albin,et al.  Proteomics Identification of Proteins in Human Cortex Using Multidimensional Separations and MALDI Tandem Mass Spectrometer*S , 2007, Molecular & Cellular Proteomics.

[130]  E Gianazza,et al.  Isoelectric focusing in immobilized pH gradients: principle, methodology and some applications. , 1982, Journal of biochemical and biophysical methods.

[131]  W. Hancock,et al.  A two step fractionation approach for plasma proteomics using immunodepletion of abundant proteins and multi-lectin affinity chromatography: Application to the analysis of obesity, diabetes, and hypertension diseases. , 2008, Journal of separation science.

[132]  Ronald J. Moore,et al.  Toward a Human Blood Serum Proteome , 2002, Molecular & Cellular Proteomics.

[133]  Richard D. Smith,et al.  Advances and Challenges in Liquid Chromatography-Mass Spectrometry-based Proteomics Profiling for Clinical Applications* , 2006, Molecular & Cellular Proteomics.

[134]  J. Bauer,et al.  Separation of plant membranes by electromigration techniques. , 1999, Journal of chromatography. B, Biomedical sciences and applications.

[135]  Elisabetta Gianazza,et al.  Protein stains for proteomic applications: Which, when, why? , 2006, Proteomics.

[136]  Steve Goodison,et al.  A comparative phosphoproteomic analysis of a human tumor metastasis model using a label‐free quantitative approach , 2010, Electrophoresis.

[137]  J. Yates,et al.  An automated multidimensional protein identification technology for shotgun proteomics. , 2001, Analytical chemistry.

[138]  P. O’Farrell High resolution two-dimensional electrophoresis of proteins. , 1975, The Journal of biological chemistry.

[139]  John R Yates,et al.  Large Scale Protein Profiling by Combination of Protein Fractionation and Multidimensional Protein Identification Technology (MudPIT)* , 2006, Molecular & Cellular Proteomics.

[140]  D. Speicher,et al.  Depletion of multiple high‐abundance proteins improves protein profiling capacities of human serum and plasma , 2005, Proteomics.

[141]  J. Rossier,et al.  Proteome analysis of human plasma and amniotic fluid by Off‐Gel™ isoelectric focusing followed by nano‐LC‐MS/MS , 2006, Electrophoresis.

[142]  L. J. Lewis,et al.  Glycosylated ovine prolactin. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[143]  L. Lomas,et al.  Protein Equalizer™ Technology : The quest for a “democratic proteome” , 2006, Proteomics.

[144]  Melinda E. Lull,et al.  Depletion of abundant proteins from non‐human primate serum for biomarker studies , 2006, Proteomics.

[145]  J. Kim,et al.  Utility of electrophoretically derived protein mass estimates as additional constraints in proteome analysis of human serum based on MS/MS analysis , 2005, Proteomics.

[146]  L. Schoofs,et al.  Gel-based versus gel-free proteomics: a review. , 2005, Combinatorial chemistry & high throughput screening.

[147]  R. Aebersold,et al.  Proteomics: the first decade and beyond , 2003, Nature Genetics.

[148]  John R Yates,et al.  Multidimensional separations for protein/peptide analysis in the post-genomic era. , 2002, BioTechniques.

[149]  Maria P. Pavlou,et al.  Nipple aspirate fluid proteome of healthy females and patients with breast cancer. , 2010, Clinical chemistry.

[150]  G. Lubec,et al.  Column chromatographic prefractionation leads to the detection of 543 different gene products in human fetal brain , 2005, Electrophoresis.

[151]  H. Zou,et al.  Immobilized Zirconium Ion Affinity Chromatography for Specific Enrichment of Phosphopeptides in Phosphoproteome Analysis*S , 2007, Molecular & Cellular Proteomics.

[152]  J. Kotzka,et al.  Combinatorial hexapeptide ligand libraries (ProteoMiner™): An innovative fractionation tool for differential quantitative clinical proteomics , 2009, Archives of physiology and biochemistry.

[153]  L. Horrocks,et al.  Purification of lipases, phospholipases and kinases by heparin-Sepharose chromatography. , 1994, Journal of chromatography. A.

[154]  David W. Speicher,et al.  Comparison of Extensive Protein Fractionation and Repetitive LC-MS/MS Analyses on Depth of Analysis for Complex Proteomes , 2009, Journal of proteome research.

[155]  D. Anderson,et al.  Gradient chromatofocusing high-performance liquid chromatography. I. Practical aspects. , 1997, Journal of chromatography. A.

[156]  Y. Ishihama,et al.  Simple on-line sample preconcentration technique for peptides based on dynamic pH junction in capillary electrophoresis-mass spectrometry. , 2007, Journal of chromatography. A.

[157]  Alexey I Nesvizhskii,et al.  Optimized peptide separation and identification for mass spectrometry based proteomics via free-flow electrophoresis. , 2006, Journal of proteome research.

[158]  R. Bacallao,et al.  Characterization of the renal cyst fluid proteome in autosomal dominant polycystic kidney disease (ADPKD) patients , 2008, Proteomics. Clinical applications.

[159]  Christine A. Miller,et al.  Efficient Fractionation and Improved Protein Identification by Peptide OFFGEL Electrophoresis*S , 2006, Molecular & Cellular Proteomics.

[160]  P. Righetti,et al.  The ProteoMiner and the FortyNiners: searching for gold nuggets in the proteomic arena. , 2008, Mass spectrometry reviews.

[161]  Richard D. Smith,et al.  Trends in mass spectrometry instrumentation for proteomics. , 2002, Trends in biotechnology.

[162]  R. Marouga,et al.  The development of the DIGE system: 2D fluorescence difference gel analysis technology , 2005, Analytical and bioanalytical chemistry.

[163]  X. Yao,et al.  Fractionation of cytosolic proteins on an immobilized heparin column. , 2003, Analytical chemistry.

[164]  D. Anderson,et al.  Effect of buffer concentration on gradient chromatofocusing performance separating protiens on a high-performance DEAE column. , 2001, Journal of chromatography. A.

[165]  W. Kolch,et al.  Capillary electrophoresis-mass spectrometry as a powerful tool in clinical diagnosis and biomarker discovery. , 2005, Mass spectrometry reviews.

[166]  T. Veenstra,et al.  On-column sample enrichment for capillary electrophoresis sheathless electrospray ionization mass spectrometry: evaluation for peptide analysis and protein identification. , 2003, Analytical chemistry.

[167]  Juri Rappsilber,et al.  Exploring the hidden human urinary proteome via ligand library beads. , 2005, Journal of proteome research.

[168]  P. Lescuyer,et al.  Comprehensive proteome analysis by chromatographic protein prefractionation , 2004, Electrophoresis.

[169]  Marcel Ottens,et al.  Selection of pH-related parameters in ion-exchange chromatography using pH-gradient operations. , 2008, Journal of chromatography. A.

[170]  J. Yates,et al.  Large-scale analysis of the yeast proteome by multidimensional protein identification technology , 2001, Nature Biotechnology.

[171]  D. Speicher,et al.  A method for global analysis of complex proteomes using sample prefractionation by solution isoelectrofocusing prior to two-dimensional electrophoresis. , 2000, Analytical biochemistry.

[172]  A. Dorner,et al.  Proteomic identification of endothelial proteins isolated in situ from atherosclerotic aorta via systemic perfusion. , 2007, Journal of proteome research.

[173]  Dorota Antos,et al.  Protein separations with induced pH gradients using cation-exchange chromatographic columns containing weak acid groups. , 2008, Journal of chromatography. A.

[174]  R. Moritz,et al.  Continuous free‐flow electrophoresis separation of cytosolic proteins from the human colon carcinoma cell line LIM 1215: A non two‐dimensional gel electrophoresis‐based proteome analysis strategy , 2001, Proteomics.

[175]  Michael A. Freitas,et al.  Liquid chromatography mass spectrometry profiling of histones. , 2007, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[176]  T. Meitinger,et al.  Improved proteome analysis of Saccharomyces cerevisiae mitochondria by free‐flow electrophoresis , 2003, Proteomics.

[177]  C. Eckerskorn,et al.  Efficient separation and analysis of peroxisomal membrane proteins using free‐flow isoelectric focusing , 2004, Electrophoresis.

[178]  Garry L Corthals,et al.  Proteomic tools for biomedicine. , 2002, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[179]  G. Anderson,et al.  High-efficiency capillary isoelectric focusing of peptides. , 2000, Analytical chemistry.

[180]  Ilan Beer,et al.  Evaluation of prefractionation methods as a preparatory step for multidimensional based chromatography of serum proteins , 2005, Proteomics.

[181]  N. Green,et al.  The properties of subunits of avidin coupled to sepharose. , 1973, The Biochemical journal.

[182]  S. Cordwell Technologies for bacterial surface proteomics. , 2006, Current opinion in microbiology.

[184]  H. Girault,et al.  Gel‐free IEF in a membrane‐sealed multicompartment cell for proteome prefractionation , 2007, Electrophoresis.

[185]  R. Bischoff,et al.  Multidimensional chromatography coupled to mass spectrometry in analysing complex proteomics samples. , 2010, Journal of separation science.

[186]  G. Irvine Size-exclusion high-performance liquid chromatography of peptides: a review , 1997 .

[187]  Mingliang Ye,et al.  Selective on-line serum peptide extraction and multidimensional separation by coupling a restricted-access material-based capillary trap column with nanoliquid chromatography-tandem mass spectrometry. , 2009, Journal of chromatography. A.

[188]  G. Elia Biotinylation reagents for the study of cell surface proteins , 2008, Proteomics.

[189]  Eugene A. Kapp,et al.  Application of 2‐D free‐flow electrophoresis/RP‐HPLC for proteomic analysis of human plasma depleted of multi high‐abundance proteins , 2005, Proteomics.

[190]  T. Kislinger,et al.  Peptide separations by on-line MudPIT compared to isoelectric focusing in an off-gel format: application to a membrane-enriched fraction from C2C12 mouse skeletal muscle cells. , 2009, Journal of proteome research.

[191]  M. Wilchek,et al.  Application of avidin-biotin technology to affinity-based separations. , 1990, Journal of chromatography.

[192]  D. Lubman,et al.  Narrow‐band fractionation of proteins from whole cell lysates using isoelectric membrane focusing and nonporous reversed‐phase separations , 2004, Electrophoresis.

[193]  Michelle A. Anderson,et al.  Comparative serum glycoproteomics using lectin selected sialic acid glycoproteins with mass spectrometric analysis: application to pancreatic cancer serum. , 2006, Journal of proteome research.

[194]  W. Barrett,et al.  Differences among techniques for high‐abundant protein depletion , 2005, Proteomics.

[195]  F. Holsboer,et al.  Mining the human cerebrospinal fluid proteome by immunodepletion and shotgun mass spectrometry , 2004, Electrophoresis.

[196]  David C Muddiman,et al.  Evaluation of protein depletion methods for the analysis of total-, phospho- and glycoproteins in lumbar cerebrospinal fluid. , 2005, Journal of proteome research.

[197]  C. Obermaier,et al.  Two‐dimensional separation of human plasma proteins using iterative free‐flow electrophoresis , 2007, Proteomics.

[198]  T. Rabilloud Two‐dimensional gel electrophoresis in proteomics: Old, old fashioned, but it still climbs up the mountains , 2002, Proteomics.

[199]  N. Anderson,et al.  Multi‐component immunoaffinity subtraction chromatography: An innovative step towards a comprehensive survey of the human plasma proteome , 2003, Proteomics.

[200]  R. Aebersold,et al.  Analysis of dilute peptide samples by capillary zone electrophoresis. , 1990, Journal of chromatography.

[201]  Ten-Yang Yen,et al.  Combining Results from Lectin Affinity Chromatography and Glycocapture Approaches Substantially Improves the Coverage of the Glycoproteome*S , 2009, Molecular & Cellular Proteomics.

[202]  A. Cifuentes,et al.  Mass distribution and focusing properties of carrier ampholytes for isoelectric focusing: I. Novel and unexpected results , 2006, Electrophoresis.

[203]  J. Rossier,et al.  Two‐stage Off‐Gel™ isoelectric focusing: Protein followed by peptide fractionation and application to proteome analysis of human plasma , 2005, Electrophoresis.

[204]  M. Baker,et al.  High‐abundance protein depletion: Comparison of methods for human plasma biomarker discovery , 2010, Electrophoresis.

[205]  V. Wasinger,et al.  Mass and charge selective protein fractionation for the differential analysis of T-cell and CD34+ stem cell proteins from cord blood. , 2010, Journal of proteomics.

[206]  Martin R Larsen,et al.  Evaluation of the impact of some experimental procedures on different phosphopeptide enrichment techniques. , 2007, Rapid communications in mass spectrometry : RCM.

[207]  J. Yates,et al.  Direct analysis of protein complexes using mass spectrometry , 1999, Nature Biotechnology.

[208]  Karl Mechtler,et al.  HPLC techniques for proteomics analysis--a short overview of latest developments. , 2006, Briefings in functional genomics & proteomics.