Elsevier S & T

The Proteome Revisited: Theory and practice of all relevant electrophoretic steps

Stoyanov, A.; Zhukov, M.; Righetti, Pier Giorgio

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Table of contents
  • Cover
  • ContentsIX
  • IntroductionV
  • Part I: Isoelectric Focussing: Fundamentals. Perspectives and Limits. Optimization of the Separation1
  • Introduction3
  • Part I.I: Isoelectric Focussing: Fundamentals7
  • Chapter 1. Electrolyte Dissociation in Water Solution. Simple Electrolytes9
  • 1.1. Introduction9
  • 1.2. Stepwise and parallel dissociation schemes for a bivalent protolyte9
  • 1.3. Relative concentration of different protolyte forms for stepwise and parallel schemes10
  • 1.4. Hydrogen ions concentration and buffer capacity12
  • 1.5. Ionisation coefficient14
  • 1.6. Isoelectric point16
  • 1.7. Mobility of protolyte molecule16
  • 1.8. Non-additive sum for buffer capacity in case of stepwise dissociation17
  • 1.9. Non-amphoteric compounds and buffer capacity in 'isoprotic state18
  • 1.10. Notations20
  • 1.11. References21
  • Chapter 2. Dissociation of Polyvalent Electrolytes23
  • 2.1. Introduction23
  • 2.2. Acid–base equilibria, macroscopic and microscopic constants24
  • 2.3. Dissociation schemes of a hybrid type27
  • 2.4. Proton transfer tautomerism29
  • 2.5. Schemes with independent dissociation30
  • 2.6. Titration curve modelling31
  • 2.7. Linderstrøm-Lang equation32
  • 2.8. Calculation of the complete set of microconstants32
  • 2.9. Relative concentration of microstates for a homopolymer (independent dissociation)34
  • 2.10. Notation36
  • 2.11. References37
  • Chapter 3. Kinetic Aspects of Acid–Base Equilibria39
  • 3.1. Introduction39
  • 3.2. Life-time of microscopic states40
  • 3.3. Relaxation of the ionic atmosphere40
  • 3.4. Modelling of the electrophoretic flux, electrophoretic mobility and conductivity42
  • 3.5. References44
  • Chapter 4. Natural pH Gradients45
  • 4.1. Introduction45
  • 4.2. Simplest examples of natural pH gradients45
  • 4.3. pH gradients created with a multi-component mixture of amphoteric compounds51
  • 4.4. References53
  • Chapter 5. Immobilised pH Gradients55
  • 5.1. Classical immobilised pH gradients created with linear density gradient55
  • 5.2. Linear pH gradients with non-linear gradients of concentration56
  • 5.3. Buffering and conductivity properties of immobilised pH gradients57
  • 5.4. Some characteristic features of electrophoresis in gel media with immobilised electric charge61
  • 5.5. Notation73
  • 5.6. References73
  • Chapter 6. Steady-State IEF75
  • 6.1. Introduction75
  • 6.2. Steady-state concentration distribution with an assumption of no sample–buffer interaction75
  • 6.3. The influence of the focussing sample on gradient properties76
  • 6.4. References80
  • Chapter 7. The Dynamics of Isoelectric Focussing81
  • 7.1. Introduction81
  • 7.2. Diffusionless approximation81
  • 7.3. The evaluation of focussing time83
  • 7.4. References84
  • Part I.II: Optimization of the Electrophoretic Separation85
  • Chapter 8. Buffering Capacity87
  • 8.1. Introduction87
  • 8.2. Buffer capacity and buffer resource87
  • 8.3. Buffer properties of solutions of proteins and nucleic acids89
  • 8.4. Biopolymers as titration agents92
  • 8.5. References93
  • Chapter 9. Optimisation of Electrophoretic Separation95
  • 9.1. Optimisation of electrophomtic separation using pH–charge relationship95
  • 9.2. Dependence of mobility on molecular mass in free solution101
  • 9.3. Isoelectric buffers. The concept of 'normalised β/λ ratio'103
  • 9.4. References104
  • Chapter 10. Two-Dimensional Methods105
  • 10.1. Two-dimensional electrophoresis105
  • 10.2. Other two-dimensional separations106
  • 10.3. Mobility versus pH curves107
  • 10.4. References108
  • Chapter 11. Limitations of the Method of Isoelectric Focussing109
  • 11.1. Introduction109
  • 11.2. Ways of generating pH gradients110
  • 11.3. Intrinsic limits of IEF114
  • 11.4. Microheterogeneity of proteins and other biopolymers116
  • 11.5. References118
  • Part II Methodology121
  • Chapter 12. Conventional Isoelectric Focussing in Gel Slabs and Capillaries and Immobilised pH Gradi123
  • 12.1. Introduction124
  • 12.2. Conventional isoelectric focussing in amphoteric buffers127
  • 12.3. Immobilised pH gradients166
  • 12.4. Capillary isoelectric focussing (cIEF)188
  • 12.5. Separation of peptides and proteins by CZE in isoelectric buffers197
  • 12.6. Conclusions207
  • 12.7. References208
  • Chapter 13. Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE)217
  • 13.1. Introduction218
  • 13.2. SDS-protein complexes: a refinement of the model219
  • 13.3. Theoretical background of Mr measurement by SDS-PAGE221
  • 13.4. Methodology225
  • 13.5. Gel casting and buffer systems242
  • 13.6. Blotting procedures261
  • 13.7. Conclusions268
  • 13.8. References269
  • Chapter 14. Two-Dimensional Maps275
  • 14.1. Introduction276
  • 14.2. Some basic methodology pertaining to 2-D PAGE280
  • 14.3. Mass spectrometry in proteomics309
  • 14.4. Informatics and proteome: interrogating databases329
  • 14.5. Pre-fractionation tools in proteome analysis351
  • 14.6. Non-denaturing protein maps368
  • 14.7. References370
  • Acknowledgements379
  • Abbreviations in Part II381
  • Subject Index383
Book details
  • Vendor Elsevier S & T
  • SKU 9780444505262
  • ISBN-13 9780080518961
  • Author Stoyanov, A.; Zhukov, M.; Righetti, Pier Giorgio
  • Category Science
  • Subject Organic

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The book deals with the theory and practice of all electrophoretic steps leading to proteome analysis, i.e. isoelectric focusing (including immobilized pH gradients), sodium dodecyl sulphate electrophoresis (SADS-PAGE) and finally two-dimensional maps. It is a reasoned collection of all modern, relevant, up-to-date methodologies leading to successful fractionation, analysis and characterization of every polypeptide spot in 2-D map analysis. It includes chapters on the most sophisticated mass spectrometry developments and it helps the reader in navigating through the most important databases in proteome analysis, including step by step tours in selected sites. Yet, this book's unique strength and feature is the fact that it combines not only practice (in common with any other book on this topic) but also theory, by giving a detailed treatment on the most advanced theoretical treatments of steady-state techniques, such as isoelectric focusing and immobilized pH gradients. A lot of this theory is newly developed and presented to the public for the first time. Thus, this book should satisfy not only the needs of every day practitioners, but also the desires of the most advanced theoreticians in the field, who will surely appreciate the novel theories presented here.
Also the methodological section contains several as yet unpublished protocols, correcting some of the existing ones and showing the pitfall and limitations of even well ingrained protocols in proteome analysis, which are here critically re-evaluated for the first time.