The Proteome Revisited

Preface. Part I: Isoelectric Focusing: Fundamentals. Perspectives and Limits. Optimization of the Separation Process. Part I.I: Isoelectric Focusing: Fundamentals. Introduction. Isoelectric Focusing: Principles and Historical Aspects. References. Electrolyte Dissociation in Water Solution.Simple Electrolytes.Stepwise and parallel dissociation schemes for a bivalent protolyte.Relative concentration of different protolyte forms for stepwise and parallel schemes. Hydrogen ions concentration and buffer capacity.Ionization coefficient. Isoelectric point.Mobility of protolyte molecule.Non additive sum for buffer capacity in case of stepwise dissociation.Non amphoteric compounds and buffer capacity in "isoprotic state".References.Dissociation of Polyvalent Electrolytes.Acid-base equilibria, macroscopic and microscopic constants.Dissociation schemes of a hybrid type.Proton transfer tautomerism.Schemes with independent dissociation. Titration curve modeling. Linderstrøm-Lang equation.Calculation of the complete set of microconstants. Relative concentration of microstates for a homopolymer (independent dissociation). References.Kinetic Aspects of Acid-base Equilibria.Life-time of microscopic states.Relaxation of the ionic atmosphere. Modeling of the electrophoretic flux, electrophoretic mobility and conductivity. References. Natural pH Gradients. Simplest examples of natural pH-gradients.pH-gradients created with a multi-component mixture of amphoteric compounds.References.Immobilized pH Gradients.Classical immobilized pH-gradients created with linear density gradient.Linear pH-gradients with non-linear gradients of concentration.Buffering and conductivity properties of immobilized pH-gradients.Some characteristic features of electrophoresis in gel media with immobilized electric charge. Method of diagonal sample application.Experimentally observed dynamics of isoelectric focussing in immobilized pH gradient gels.Low-molecular mass ion adsorption on weak ion exchanger.References.Steady-State IEF. Steady state concentration distribution with an assumption of no sample-buffer interaction.The influence of the focussing sample on gradient properties.Low sample concentrations.High sample concentrations.References.The Dynamics of Isoelectric Focusing.Diffusionless approximation.The evaluation of focusing time.References.Part I.II: Optimization of the Elctrophoretic Separation. Buffering Capacity. Buffer capacity and buffer resource.Buffer properties of solutions of proteins and nucleic acids.Biopolymers as titration agents.References. Optimization of Electrophoretic Separation. Optimization of electrophoretic separation using pH-charge relationship.Calculation of mobility vs. pH.Relative charge difference for two components to be separated.Dependence of mobility on molecular mass in free solution.Isoelectric buffers. The concept of "Normalized b/l ratio".References. Two Dimensional Methods. Two-dimensional electrophoresis. Other two-dimensional separations.Mobility versus pH curves.References. Limitations of the Method of Isoelectric Focussing. Ways of generating pH gradients.Natural and artificial pH gradients.Thermal pH gradients.External temperature field.Thermal pH gradients created by Joule heat dissipation in electrophoretic chamber with non-constant cross section. Gradient in dielectric constant.Dielectric constant influence on buffer dissociation constant.Gradient of electric field coupled with dielectric constant gradient.Intrinsic limits of IEF.Microheterogeneity of proteins and other biopolymers.pH shift due to single modifications of ionogenic groups. Multiple one-type modifications. References.Part II: Methodology Conventional Isoelectric Focusing in Gel Slabs and Capillaries and Immobilized pH Gradients. Introductionbrief historical survey.Conventional isoelectric focusing in amphoteric buffers.General considerations.The basic method.Applications and limitations.Specific advantages.Carrier ampholytes.Equipment.Electrophoretic equipment.Polymerization cassette.The polyacrylamide matrix.Reagents.Gel formulations.Choice of carrier ampholytes.Gel preparation and electrophoresis.Assembling the mould.Filling the mould.Gel polymerization.Sample loading and electrophoresis.General protein staining.Coomassie Blue G-250. Coomassie Blue R-250/CuSO4. Coomassie Blue R-250/sulphosalicylic acid.Silver stain. Coomassie Blue G-250/urea/perchloric acid.Fluorescence protein detection.Specific protein detection methods.Quantitation of the focused bands.Troubleshooting.Waviness of bands near the anode.Burning along the cathodic strip.pH gradient different from expected. Sample precipitation at the application point.Some typical applications of IEF.Examples of some fractionations.Artefacts or not?Immobilized pH gradients.General considerations.The problems of conventional IEF.The Immobiline matrix.Narrow and ultranarrow pH gradients.Extended pH gradients: general rules for their generation and optimization.Non-linear, extended pH gradients.Extremely alkaline pH gradients.IPG Methodology.Casting an Immobiline gel.Reswelling dry Immobiline gels.Electrophoresis.Staining and pH measurements.Storage of the Immobiline chemicals.Mixed bed, CA-IPG gels.Trouble-shooting.Some analytical results with IPGs.Capillary isoelectric focusing (cIEF).General considerations.cIEF methodology.General guidelines for cIEF. Increasing the resolution by altering the slope of the pH gradient.On the problem of protein solubility at their pI.Assessment of pH gradient and pI values in cIEF.Separation of peptides and proteins by CZE in isoelectric buffers.General properties of amphoteric, isoelectric buffers. Examples of some separations of proteins in isoelectric buffers.Troubleshooting for CZE in isoelectric buffers. Conclusions.References.Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE). Introduction.SDS-protein complexes: a refinement of the model.Theoretical background of Mr measurement by SDS-PAGE.Methodology.Purity and detection of SDS.Molecular mass markers.Prelabelling with dyes or fluorescent markers.Post-electrophoretic detection. Non-diamine, silver nitrate stain.Colloidal staining."Hot" Coomassie staining.Turbidimetric protein detection (negative stain). Negative metal stains.Fluorescent detection with SYPRO dyes.Possible sources of artefactual protein modification.On the use and properties of surfactants.The use of surfactants other than SDS.Anomalous behaviour.Gel casting and buffer systems.Sample pretreatment.The standard method using continuous buffers.The composition of gels and buffers.Use of discontinuous buffers.The method of Neville.The method of Laemmli.Porosity gradient gels.Peptide mapping by SDS-PAGE.SDS-PAGE in photopolymerized gels. Blotting procedures.Capillary and electrophoretic transfer.Detection systems after blotting.Conclusions.References.Two-Dimensional Maps.Introduction.The early days and the evolution of 2-D PAGE. A glimpse at modern times.Some basic methodology pertaining to 2-D PAGE.Methods of cell disruption.Proteolytic attack during cell disruption.Precipitation procedures.Removal of interfering substances.Solubilization cocktail.Sample application.Sequential sample extraction. Mass spectrometry in proteomics. MALDI-TOF mass spectrometry.ESI mass spectrometry.Nanoelectrospray mass spectrometry.Mass spectrometry for quantitative proteomics.Labelling before extraction.Labelling after extraction.Multidimensional chromatography coupled to mass spectrometry. Informatics and Proteome: interrogating databases.An example of navigation on 2-D maps sites.The SWISS-PROT database.TrEMBL: a supplement to SWISS-PROT. The SWISS-2DPAGE database. Database searching via mass-spectrometric information. Pre-fractionation tools in proteome analysis. Sample pre-fractionation via different chromatographic approaches.Sample pre-fractionation via multicompartment electrolyzers with Immobiline membranes.Non-denaturing protein maps.References.Subject Index.