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El. knyga: Proteome Revisited: Theory and Practice of all Relevant Electrophoretic Steps

(Institute of Physical Chemistry, Russian Academy of Sciences, Moscow, Russia), (Department of Mechanics and Mathematics, Rostov State University, Rostov-na-don, Russia), (Miles Gloriosus Academy, Milano, Italy)
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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Also the methodological section contains several as yet unpublished protocols, correcting some of the existing ones and showing the pitfall and limitations of even well ingrained protocols in proteome analysis, which are here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.

Recenzijos

"...this is a valuable book that should be read by all those working in proteomics and interested in understanding the electrophoretic principles behind 'classical proteomics." --Proteomics

Introduction v
Part I Isoelectric Focussing: Fundamentals. Perspectives and Limits. Optimization of the Separation Process
Introduction
3(6)
Part I.I Isoelectric Focussing: Fundamentals
Electrolyte Dissociation in Water Solution. Simple Electrolytes
9(14)
Introduction
9(1)
Stepwise and parallel dissociation schemes for a bivalent protolyte
9(1)
Relative concentration of different protolyte forms for stepwise and parallel schemes
10(2)
Hydrogen ions concentration and buffer capacity
12(2)
Ionisation coefficient
14(2)
Isoelectric point
16(1)
Mobility of protolyte molecule
16(1)
Non-additive sum for buffer capacity in case of stepwise dissociation
17(1)
Non-amphoteric compounds and buffer capacity in `isoprotic state'
18(2)
Notations
20(1)
References
21(2)
Dissociation of Polyvalent Electrolytes
23(16)
Introduction
23(1)
Acid-base equilibria, macroscopic and microscopic constants
24(3)
Dissociation schemes of a hybrid type
27(2)
Proton transfer tautomerism
29(1)
Schemes with independent dissociation
30(1)
Titration curve modelling
31(1)
Linderstrøm-Lang equation
32(1)
Calculation of the complete set of microconstants
32(2)
Relative concentration of microstates for a homopolymer (independent dissociation)
34(2)
Notation
36(1)
References
37(2)
Kinetic Aspects of Acid-Base Equilibria
39(6)
Introduction
39(1)
Life-time of microscopic states
40(1)
Relaxation of the ionic atmosphere
40(2)
Modelling of the electrophoretic flux, electrophoretic mobility and conductivity
42(2)
References
44(1)
Natural pH Gradients
45(10)
Introduction
45(1)
Simplest examples of natural pH gradients
45(6)
pH gradients created with a multi-component mixture of atmophteric compounds
51(2)
References
53(2)
Immobilised pH Gradients
55(20)
Classical immobilised pH gradients created with linear density gradient
55(1)
Linear pH gradients with non-linear gradients of concentration
56(1)
Buffering and conductivity properties of immobilised pH gradients
57(4)
Some characteristic features of electrophoresis in gel media with immobilised electric charge
61(12)
Method of diagonal sample application
61(1)
Experimentally observed dynamics of isoelectric focussing in immobilised pH gradient gels
62(4)
Low-molecular mass ion adsorption on weak ion exchanger
66(7)
Notation
73(1)
References
73(2)
Steady-State IEF
75(6)
Introduction
75(1)
Steady-state concentration distribution with an assumption of no sample-buffer interaction
75(1)
The influence of the focussing sample on gradient properties
76(4)
Low sample concentrations
76(3)
High sample concentrations
79(1)
References
80(1)
The Dynamics of Isoelectric Focussing
81(6)
Introduction
81(1)
Diffusionless approximation
81(2)
The evaluation of focussing time
83(1)
References
84(3)
Part I.II Optimization of the Electrophoretic Separation
Buffering Capacity
87(8)
Introduction
87(1)
Buffer capacity and buffer resource
87(2)
Buffer properties of solutions of proteins and nucleic acids
89(3)
Biopolymers as titration agents
92(1)
References
93(2)
Optimisation of Electrophoretic Separation
95(10)
Optimisation of electrophoretic separation using pH-charge relationship
95(6)
Calculation of mobility vs. pH
95(1)
Relative charge difference for two components to be separated
96(5)
Dependence of mobility on molecular mass in free solution
101(2)
Isoelectric buffers. The concept of `normalised β/λ ratio'
103(1)
References
104(1)
Two-Dimensional Methods
105(4)
Two-dimensional electrophoresis
105(1)
Other two-dimensional separations
106(1)
Mobility versus pH curves
107(1)
References
108(1)
Limitations of the Method of Isoelectric Focussing
109(14)
Introduction
109(1)
Ways of generating pH gradients
110(4)
Natural and artificial pH gradients
110(1)
Thermal pH gradients
110(1)
External temperature field
110(3)
Thermal pH gradients created by Joule heat dissipation in an electrophoretic chamber with a non-constant cross-section
113(1)
Gradient in dielectric constant
113(1)
Dielectric constant influence on buffer dissociation constant
113(1)
Gradient of electric field coupled with dielectric constant gradient
114(1)
Intrinsic limits of IEF
114(2)
Microheterogeneity of proteins and other biopolymers
116(2)
pH shift due to single modifications of ionogenic groups
116(1)
Multiple one-type modifications
117(1)
References
118(5)
Part II Methodology
Conventional Isoelectric Focussing in Gel Slabs and Capillaries and Immobilised pH Gradients
123(94)
Introduction
124(3)
A brief historical survey
125(2)
Conventional isoelectric focussing in amphoteric buffers
127(39)
General considerations
127(1)
The basic method
128(1)
Applications and limitations
129(1)
Specific advantages
130(1)
Carrier ampholytes
130(2)
Equipment
132(1)
Electrophoretic equipment
132(1)
Electrophoretic chamber
132(1)
Power supply
132(1)
Thermostatic unit
132(1)
Polymerisation cassette
133(1)
Gel supporting plate
133(1)
Spacer
134(1)
Cover plate
135(1)
Clamps
135(1)
The Polyacrylamide gel matrix
136(1)
Reagents
136(2)
Gel formulations
138(1)
Choice of carrier ampholytes
138(3)
Gel preparation and electrophoresis
141(1)
Assembling the gel mould
142(1)
Filling the mould
142(2)
Gel polymerisation
144(1)
Sample loading and electrophoresis
144(4)
General protein staining
148(3)
Micellar Coomassie Blue G-250
151(1)
Coomassie Blue R-250/CuSo4
151(1)
Coomassie Blue R-250/sulphosalicylic acid
152(1)
Silver stain
152(1)
Coomassie Blue G-250/urea/perchloric acid
153(1)
Fluorescence protein detection
153(1)
Specific protein detection methods
154(1)
Quantitation of the focussed bands
154(1)
Troubleshooting
155(1)
Waviness of bands near the anode
155(1)
Burning along the cathodic strip
155(1)
pH gradients different from expected
155(1)
Sample precipitation at the application point
155(1)
Some typical applications of IEF
156(1)
Examples of some fractionations
157(5)
Artefacts or not?
162(4)
Immobilised pH gradients
166(22)
General considerations
166(1)
The problems of conventional IEF
166(1)
The Immobiline matrix
167(3)
Narrow and ultra narrow pH gradients
170(4)
Extended pH gradients: general rules for their generation and optimisation
174(1)
Non-linear, extended pH gradients
175(2)
Extremely alkaline pH gradients
177(1)
IPG methodology
178(1)
Casting an Immobiline gel
178(4)
Reswelling dry Immobiline gels
182(1)
Electrophoresis
183(1)
Staining and pH measurements
184(1)
Storage of the Immobiline chemicals
184(1)
Mixed-bed, CA-IPG gels
185(1)
Troubleshooting
186(1)
Some analytical results with IPGs
187(1)
Capillary isoelectric focussing (cIEF)
188(9)
General considerations
188(2)
cIEF methodology
190(1)
General guidelines for cIEF
190(1)
Increasing the resolution by altering the slope of the pH gradient
191(3)
On the problem of protein solubility at their pI
194(2)
Assessment of pH gradients and pI values in cIEF
196(1)
Separation of peptides and proteins by CZE in isoelectric buffers
197(10)
General properties of amphoteric, isoelectric buffers
198(3)
Examples of some separations of proteins in isoelectric buffers
201(3)
Troubleshooting for CZE in isoelectric buffers
204(3)
Conclusions
207(1)
References
208(9)
Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE)
217(58)
Introduction
218(1)
SDS-protein complexes: a refinement of the model
219(2)
Theoretical background of Mr measurement by SDS-PAGE
221(4)
Methodology
225(17)
Purity and detection of SDS
225(1)
Molecular mass markers
225(1)
Prelabelling with dyes or fluorescent markers
226(2)
Post-electrophoretic detection
228(1)
Non-diamine, silver nitrate stain
229(1)
Colloidal staining
230(1)
`Hot' Coomassie staining
231(1)
Turbidimetric protein detection (negative stain)
231(1)
Negative metal stains
232(1)
Fluorescent detection with SYPRO dyes
233(2)
Possible sources of artefactual protein modification
235(1)
On the use and properties of surfactants
236(4)
The use of surfactants other than SDS
240(2)
Anomalous behaviour
242(1)
Gel casting and buffer systems
242(19)
Sample pretreatment
243(2)
The standard method using continuous buffers
245(1)
The composition of gels and buffers
246(1)
Use of discontinuous buffers
247(2)
The method of Neville
249(1)
The method of Laemmli
250(1)
Porosity gradient gels
251(4)
Peptide mapping by SDS-PAGE
255(3)
SDS-PAGE in photopolymerised gels
258(3)
Blotting procedures
261(7)
Capillary and electrophoretic transfer
262(2)
Detection systems after blotting
264(4)
Conclusions
268(1)
References
269(6)
Two-Dimensional Maps
275(104)
Introduction
276(4)
The early days and the evolution of 2-D PAGE
277(1)
A glimpse at modern times
278(2)
Some basic methodology pertaining to 2-D PAGE
280(29)
Methods of cell disruption
282(1)
Proteolytic attack during cell disruption
283(3)
Precipitation procedures
286(1)
Removal of interfering substances
287(6)
Solubilisation cocktail
293(7)
Sample application
300(7)
Sequential sample extraction
307(2)
Mass spectrometry in proteomics
309(20)
MALDI-TOF mass spectrometry
311(7)
ESI mass spectrometry
318(3)
Nanoelectrospray mass spectrometry
321(2)
Mass spectrometry for quantitative proteomics
323(1)
Labelling before extraction
324(1)
Labelling after extraction
325(2)
Multidimensional chromatography coupled to mass spectrometry
327(2)
Informatics and proteome: interrogating databases
329(22)
An example of navigation on 2-D map sites
331(6)
The SWISS-PROT database
337(1)
TrEMBL: a supplement to SWISS-PROT
338(1)
The SWISS-2DPAGE database
338(4)
Database searching via mass-spectrometric information
342(9)
Pre-fractionation tools in proteome analysis
351(17)
Sample pre-fractionation via different chromatographic approaches
352(6)
Sample pre-fraction via multicompartment electrolysers with Immobiline membranes
358(10)
Non-denaturing protein maps
368(2)
References
370(9)
Acknowledgements 379(2)
Abbreviations in Part II 381(2)
Subject Index 383