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E-raamat: Separation Methods In Proteomics

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  • Keel: eng
  • ISBN-13: 9781040199909
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Driven by the widespread growth of proteomic practices, protein separation techniques have been refined to minimize variability, optimize particular applications, and adapt to user preferences in the analysis of proteins.

Separation Methods in Proteomics provides a comprehensive examination of all major separation techniques for proteomics research.

Written as a compilation of hands-on methods exemplified by the work of several recognized leaders in the field, this book may serve as a guide for selection of the optimal separation strategies to solve particular biological problems.

Recent progress in the development of robust analytical techniques and instrumentation has created the need for good quality biological samples that are subject to analysis. Emphasizing the importance of sample preparation, the book explains how proteomes can be divided into smaller, less complicated subproteomes" for individual analysis. It also highlights several hybrid approaches that take into account protein interactions.

Including applications of the separation methods currently employed in proteomic analyses for both clinical and basic research, Separation Methods in Proteomics contains practical information that can enhance the current and future endeavors of scientists in proteomics, genomics, transcriptomics, biomarker discovery, and drug discovery.
Part I Sample Preparation
Applications of Pressure Cycling Technology (PCT) in Proteomics
3(16)
Feng Tao
James Behnke
Chunqin Li
Calvin Saravis
Richard T. Schumacher
Nathan P. Lawrence
Introduction
3(2)
Sample Preparation by the PCT SPS Compared to Traditional Methods
5(3)
Potential Mechanisms of Action of PCT
8(1)
PCT and Protein Stability
9(1)
PCT and the Role of Extraction Buffers
10(1)
Examples of Protein Extraction Using the PCT SPS
11(4)
Conclusion
15(4)
References
17(2)
Applications of Ion-Exchange (IEX) Chromatography to Reduce Sample Complexity Prior to Two-Dimensional Gel Electrophoresis (2DE)
19(10)
Malcolm G. Pluskal
Eva Golenko
Mary F. Lopez
Introduction
19(1)
IEX Chromatography Basics
20(2)
Theory
20(1)
IEX Chromatography Media Available
20(1)
Illustration of IEX Chromatography Separation Correlated with 2DE
21(1)
Applications of IEX Chromatography Prior to 2DE Analysis
22(5)
Fractionation of Human Plasma
22(1)
Impact on IEX Prefractionation on Resolution in 2DE
23(4)
Summary
27(2)
References
27(2)
Separations in Proteomics: Use of Camelid Antibody Fragments in the Depletion and Enrichment of Human Plasma Proteins for Proteomics Applications
29(12)
Mark ten Haaft
Pim Hermans
Bruce Dawson
Introduction
30(1)
Heavy-Chain Antibodies from the Camelid Family
31(1)
Roadmap to Llama Antibodies
31(7)
Stage 1: Immunization of Llamas and Creation of the VHH Library
32(1)
Stage 2: Screening for Antigen-Binding VHH Fragments
32(1)
Stage 2: Screening for the Ultimate Ligand
32(1)
Stage 3: Ligand Production
33(1)
Stage 3: Coupling to Matrices
33(2)
Stage 3: Depletion of HSA and Human IgG from Human Plasma
35(3)
Conclusions
38(3)
Acknowledgments
38(1)
References
38(3)
Novel Plasma Protein Separation Strategy Using Multiple Avian IgY Antibodies for Proteomic Analysis
41(22)
Sun W. Tam
Lei Huang
Douglas Hinerfeld
David Innamorati
Xiangming Fang
Wei-Wei Zhang
John Pirro
Jerald S. Feitelson
Introduction
42(2)
Materials and Methods
44(2)
Coupling of Affinity-Purified IgY to UltraLink® Hydrazide Gel
44(1)
Spin Column Method for Removal of Serum and Plasma Abundant Protein
44(1)
Albumin Depletion with Liquid Chromatography Workstation
44(1)
One-Dimensional Electrophoresis
45(1)
Two-Dimensional Electrophoresis
45(1)
Protein Digestion and MALDI Analysis
45(1)
Bioinformatic Database Search
45(1)
Results
46(7)
Cibacron Blue Depletion of Rat Albumin Removed Many Nontarget Proteins
46(1)
Structural Comparisons of IgG and IgY and Their Covalent Coupling to Microbead Carriers
46(2)
IgY Gels Have High Capacity and Specificity
48(1)
Anti-HSA IgY Gels Have Excellent Cross-Reactivity to Various Mammalian Albumins
48(1)
IgYs Cross-React and Are Also Effective at Separating IgG, Fibrinogen, and Transferrin from Human and Rat Plasma
49(1)
Six Abundant Plasma Proteins Are Effectively Removed by MIXED6 IgY Gels
50(2)
High-Throughput Proteomic Sample Processing Is Facilitated by Use of FPLC Techniques
52(1)
Discussion
53(10)
Acknowledgments
60(1)
References
60(3)
Immunoaffinity Depletion of High-Abundant Proteins for Proteomic Sample Preparation
63(18)
Nina Zolotarjova
Barry Boyes
James Martosella
Liang-Sheng Yang
Gordon Nicol
Kelly Zhang
Cory Szafranski
Jerome Bailey
Introduction
64(2)
Methods
66(5)
Immunodepletion Column
66(1)
Sample Collection and Processing
67(1)
Chromatographic Set-Up
67(1)
Immunoaffinity Separation
67(1)
Analysis of Flow-Through Fractions by Enzyme-Linked Immunosorbent Assay (ELISA)
68(1)
Processing of Depleted Serum and Bound Fractions for Down-Stream Analysis
69(1)
One-Dimensional (1D) and Two-Dimensional (2D) Gel Electrophoresis
69(1)
Liquid Chromatography--Tandem Mass Spectrometry (LC-MS/MS)
69(1)
Cibacron Blue Column
70(1)
Analysis of Cibacron Blue Bound Fraction
70(1)
Analysis of Cibacron Blue Flow-Through Fraction
71(1)
Results
71(6)
Immunoaffinity Column Performance and Reproducibility
71(1)
Completeness of the Depletion of High-Abundant Proteins
71(2)
Specificity of the Immunodepletion
73(4)
Conclusions
77(4)
References
78(3)
Isolation of Plasma Membrane Proteins for Proteomic Analysis
81(8)
Catherine Fenselau
Amir Rahbar
Introduction
81(1)
Preparation of the Plasma Membrane Fraction
82(2)
Materials
82(1)
Cell Suspensions
82(1)
Cells Growing in Monolayers
83(1)
Proteomics Analyses
84(1)
Conclusions
85(4)
Acknowledgments
86(1)
References
86(3)
New Ultrafiltration and Solid Phase Extraction Techniques Improve Serum Peptide Detection
89(18)
Elena Chernokalskaya
Sara Gutierrez
Aldo M. Pitt
Alexander V. Lazarev
Jack T. Leonard
Introduction
89(2)
Methods
91(2)
Preparation of Serum Peptides by UF and SPE
91(1)
Preparation of Serum Peptides by Acetonitrile Precipitation
92(1)
Peptide Analysis by Mass Spectrometry
93(1)
Two-Dimensional (2D) Gel Electrophoresis
93(1)
Results
93(4)
Conclusion
97(10)
References
98(9)
Part II Sample Prefractionation and Analysis
Tools for Sample Preparation and Prefractionation in Two-Dimensional (2D) Gel Electrophoresis
107(28)
Anton Posch
Aran Paulus
Mary Grace Brubacher
Introduction
108(1)
Sample Preparation Basics
109(10)
Cell Disruption Methods
109(2)
Sample and Lysis Buffer Constituents
111(1)
Chaotropic Agents
111(1)
Detergents
112(1)
Reducing Agents
113(1)
Carrier Ampholytes
114(1)
General Solubilization Cocktails and Sample Preparation Guidelines
114(1)
Technologies for Protein Sample Preparation
115(1)
Solution Chemistry
115(2)
Chromatographic Methods
117(1)
Electrophoretic Methods
118(1)
General-Purpose Cleanup for Improved Resolution and Reproducibility
119(3)
Removal of Interfering Contaminants
119(1)
Removal of Salt
120(1)
Nucleic Acids
120(1)
Polysaccharides
121(1)
Lipids
121(1)
Insoluble Material
121(1)
Removal of Disulfide Bonds
122(1)
Fractionation: The Quest for Low-Abundance Proteins
122(5)
Fractionation by Subcellular Location
123(1)
Fractionation by Differential Solubility
123(1)
Fractionation by Protein Size
124(1)
Fractionation by Charge and pI
124(1)
Rotofor System
125(1)
Multicompartment Electrolyzers
126(1)
Off-Gel IEF in Multicompartment Devices
127(1)
Continuous Free-Flow Electrophoresis (FFE)
127(1)
The Gradiflow System
127(1)
Concluding Remarks
127(8)
References
128(7)
Optic Nerve Fractionation for Proteomics
135(22)
Sanjoy K. Bhattacharya
John S. Crabb
Suresh P. Annangudi
Karen A. West
Xiaorong Gu
Jian Sun
Vera L. Bonilha
Gary B. Smejkal
Karen Shadrach
Joe G. Hollyfield
John W. Crabb
Introduction
135(1)
Materials and Methods
136(2)
Human Tissue Procurement
136(1)
Optic Nerve Sample Preparation
136(1)
Acetone Precipitation
137(1)
Solution-Phase IEF
137(1)
2D Gel Electrophoresis
138(1)
Protein Identification
138(1)
Results
138(14)
Extraction of Protein from Optic Nerve
138(3)
Optic Nerve IEF Using the Multicompartment Electrolyzer
141(1)
Identification of Optic Nerve Proteins following MCE Fractionation
142(10)
Discussion
152(5)
Acknowledgments
153(1)
References
153(4)
Fractionation of Retina for Proteomic Analyses
157(30)
Sanjoy K. Bhattacharya
Karen A. West
Xiaorong Gu
John S. Crabb
Kutralanathan Renganathan
Zhiping Wu
Jian Sun
John W. Crabb
Introduction
157(1)
Materials and Methods
158(2)
Bovine Retina Sample Preparation
158(1)
Western Blot Analysis and 1D Electrophoresis
159(1)
Solution-State IEF
159(1)
Acetone Precipitation
159(1)
Protein Identification
159(1)
Results
160(21)
Comparison of Detergent Extracts of Bovine Retina
160(1)
Retina Fractionation by Solution-State IEF and 1D Page
160(3)
Protein Identification
163(18)
Discussion
181(6)
Acknowledgments
183(1)
References
183(4)
Reducing Protein Sample Complexity with Free Flow Electrophoresis (FFE)
187(20)
Askar Kuchumov
Gerhard Weber
Christoph Eckerskorn
Introduction
187(1)
Principle of Free Flow Electrophoresis
188(3)
Practical Considerations
191(3)
Separation Buffers
191(1)
Making an FFE Run
192(1)
Sample Preparation
192(1)
Anticipated Results
193(1)
Post-FFE Treatment of Fractions
193(1)
Applications
194(8)
Liver Proteome Fractionation
194(2)
Plasma Proteome Fractionation
196(6)
Concluding Remarks
202(5)
References
203(4)
Part III Applications of Electrophoresis in Proteomics
Destreaking Strategies for Two-Dimensional Electrophoresis
207(12)
Fengju Bai
Sheng Liu
Frank A. Witzmann
Two-dimensional Gel Electrophoresis (2DE) and Basic End Streaks
207(1)
Chemical Nature of Protein Streaking
208(1)
Destreaking Techniques
208(4)
Recent Advances in Destreaking Techniques
212(7)
References
216(3)
Proteomic Approaches to the Study of Rheumatoid Arthritis
219(16)
Mikaela Antonovici
Kumar Dasuri
Hani El-Gabalawy
John A. Wilkins
Introduction
219(1)
Rheumatoid Arthritis
220(1)
Methods and Materials
221(2)
Clinical Samples and Cell Culture
221(1)
Two-Dimensional Polyacrylamide Gel Electrophoresis (2D Page) Analysis of FLS
221(1)
Mass Spectrometry (MS) and Protein Identification
222(1)
MCE Fractionation of Synovial Fluid
222(1)
Chromatographic Separation
222(1)
SDS-Page of MCE Fractions
223(1)
MS
223(1)
Protein Identification
223(1)
Results and Discussion
223(8)
Analysis of FLS
223(2)
Analysis of Synovial Fluid
225(6)
Conclusion
231(4)
References
231(4)
Immunoglobulin Patterns in Health and Disease
235(34)
Ingrid Miller
Marcia Goldfarb
Introduction
236(1)
Immunoglobulins and Disorders
236(4)
Humoral Immune System
236(1)
Disorders
237(1)
Multiple Myeloma
237(1)
Waldenstrom's Macroglobulinemia
237(1)
Solitary Plasmacytoma
238(1)
Amyloidosis
239(1)
Heavy-Chain Diseases
239(1)
Monoclonal Gammopathy of Undetermined Significance
239(1)
Immunodeficiency
240(1)
Methods to Investigate Immunoglobulin Patterns (as Exemplified for Serum)
240(2)
One-Dimensional Separations
240(1)
2DE
241(1)
Specific Detection
242(1)
Serum
242(10)
Physiological Patterns
242(2)
Pathological Conditions
244(1)
Monoclonal Gammopathies
244(4)
Biclonal and Oligoclonal Gammopathies
248(2)
Other Gammopathies
250(2)
Urine
252(2)
Patterns of Healthy Individuals
252(1)
Pathological Patterns
252(2)
Single-Chain Disregulations (in Serum and Urine)
254(3)
Bence Jones Proteins
254(3)
Heavy-Chain Disease
257(1)
Other Body Fluids
257(2)
Cerebrospinal Fluid (CSF)
257(1)
Saliva, Tears, and Milk
258(1)
Effusions and Lavage Fluids
259(1)
Conclusions and Perspectives
259(10)
References
259(10)
Difference Gel Electrophoresis (DIGE)
269(18)
Mustafa Unlu
Jonathan Minden
Introduction
270(3)
Of Proteomes and Gels
270(1)
DIGE
271(1)
Of Proteomes and Dynamic Range of Expression
272(1)
Good Night, Sweet Gel?
273(1)
Usage Guide
273(4)
General Notes
274(1)
Recipes, Apparatus, and Chemicals for Sample Solubilization and Labeling
274(1)
Lysis Buffer
274(1)
Labeling Solution
274(1)
Quenching Solution
275(1)
IEF
275(1)
Rehydration Buffer
275(1)
Equilibration Buffer I
275(1)
Equilibration Buffer II
275(1)
Sodium Dodecyl Sulfate--Polyacrylamide Gel Electrophoresis (SDS-Page)
275(1)
4 X Resolving Gel Buffer
276(1)
30% Monomer Solution
276(1)
4 X Stacking Gel Buffer
276(1)
Light Gradient Gel Solution
276(1)
Heavy Gradient Gel Solution
276(1)
Stacking Gel Solution
277(1)
Imager Specifications
277(1)
Destain Solution
277(1)
Method
277(10)
Sample Solubilization
277(1)
Introduction and General Notes
277(1)
Drosophila Embryos
278(1)
S. Cerevisiae
279(1)
Sample Labeling
279(1)
IEF
280(1)
Introduction and General Notes
280(1)
Rehydration
280(1)
Setting Up and Running the First Dimension
280(1)
Equilibration of IEF Gels
281(1)
SDS-PAGE
281(1)
Assembling the Gel Cassettes
281(1)
Pouring the 10--15% Gradient Gels
282(1)
Setting Up and Running the Second Dimension
282(1)
Image Acquisition and Analysis
282(1)
Introduction and General Notes
282(1)
Image Acquisition
283(1)
Image Analysis
284(1)
References
284(3)
Principles and Challenges of Basic Protein Separation by Two-Dimensional (2D) Electrophoresis
287(14)
Anton Posch
Aran Paulus
Mary Grace Brubacher
Introduction
287(1)
Principles of IPGs
288(3)
Chemistry of IPGs
288(1)
Casting of IPG Gels
289(1)
Types of IPG Gels for 2D Electrophoresis
289(1)
Sample Application
289(1)
Cup Loading
290(1)
In-Gel Rehydration
290(1)
Overcoming Difficulties with Basic Protein Separations
291(5)
Instability of the Acrylamide Matrix
292(1)
Running Conditions for Basic IPG Strips
293(2)
Reduction and Alkylation Prior to 2D Electrophoresis
295(1)
Concluding Remarks
296(5)
References
297(4)
Multidimensional Separation of Membrane Proteins
301(20)
Susan Francis-McIntyre
Simon J. Gaskell
Introduction
301(2)
Integral Membrane Proteins
302(1)
Membrane-Associated Proteins
302(1)
The Need for Membrane Proteomics
302(1)
Classical Two-Dimensional Electrophoresis (2DE) Based Methods of Membrane Protein Isolation and Characterization
303(5)
Detergent Extraction Based Approaches
304(2)
Sequential Extraction Based Techniques
306(1)
Organic Solvent Based Extraction
307(1)
Membrane Stripping Procedures
308(1)
Liquid Chromatography Coupled with Mass Spectrometry Approaches
308(5)
1D LC-MS/MS Analysis Techniques
309(3)
Shotgun Proteomics: 2D LC-MS/MS Approaches
312(1)
Fungal Proteomics Examples
313(3)
Conclusions
316(5)
Acknowledgments
316(1)
References
317(4)
Structural Approaches in Glycoproteomics
321(24)
Heidi Geiser
Cristina Silvescu
Vernon Reinhold
The Developing Field of Glycoproteomics
321(4)
Significance of Molecular Glycosylation
322(1)
Basic Glycan Structures
322(3)
Separation by Two-Dimensional Gel Electrophoresis
325(1)
Limitations
325(1)
Methods
326(1)
Glycoprotein Detection on 2D Gels
326(1)
Methods
326(1)
Glycoprotein Analysis
327(4)
Protein Sequencing
327(1)
MS Profiles of Intact Glycoproteins: Top Down
328(1)
MS Profiles of Intact Glycoproteins: Bottom Up
329(1)
Consensus Site Methods
330(1)
Glycan Release Techniques
331(2)
Methods
332(1)
Enzymatic Release
332(1)
Discussion
333(1)
Sequencing Released Glycans
333(12)
The Details of Glycan Structure by MSn
334(5)
References
339(6)
Enrichment and Analysis of Glycoproteins in the Proteome
345(18)
Nicole L. Wilson
Niclas G. Karlsson
Nicolle H. Packer
Introduction
345(1)
Lectin Affinity Chromatography
346(1)
Boronate Affinity Chromatography
346(1)
Methods
347(5)
Lectin Affinity Chromatography
347(1)
Boronate Affinity Chromatography
348(1)
2D Page
348(1)
Protein and Glycoprotein Staining
349(1)
Proteins
349(1)
Glycoproteins
350(1)
Glycosylation Analysis
350(1)
N-Linked Oligosaccharides
351(1)
O-Linked Oligosaccharides
351(1)
LC-ESI-MS of Oligosaccharides
351(1)
Results and Discussion
352(11)
References
358(5)
Part IV Applications of High-Performance Liquid Chromatography
Proteomic Analyses Using High-Efficiency Separations and Accurate Mass Measurements
363(24)
Jon M. Jacobs
Richard D. Smith
Introduction
363(1)
High-Throughput Approach for Global Proteomic Measurements
364(2)
Essential Components for Effective LC-MS Proteomic Analysis
366(10)
Electrospray Ionization Efficiency
367(2)
High-Efficiency LC Separations
369(2)
Expanded Dynamic Range for MS
371(1)
Data Processing Components
371(2)
Data Reduction Processes
373(1)
Peptide Identifications Using Accurate Mass and Time Measurements
373(3)
Quantitation Strategies
376(3)
18O Stable-Isotope Labeling Techniques
376(2)
``Label-free'' Quantitation Using Intensity Information
378(1)
Recent Applications
379(3)
Conclusions
382(5)
Acknowledgments
382(1)
References
383(4)
Middle-Out Proteomics: Incorporating Multidimensional Protein Fractionation and Intact Protein Mass Analysis as Elements of a Proteomic Workflow
387(32)
Scott J. Berger
Kevin M. Millea
Ira S. Krull
Steven A. Cohen
Introduction
388(4)
Proteomics Strategies
388(2)
Protein Fractionation
390(1)
Combining Top-Down and Bottom-Up Proteomics
391(1)
Methods
392(2)
Ribosomal Protein Analysis
392(1)
Escherichia coli Analysis
393(1)
AutoME Data Analysis
394(1)
Results and Discussion
394(16)
Overview
394(1)
From the Top Down
395(1)
Mass Analysis of Intact Proteins
395(2)
Fractionation at the Intact Protein Level
397(3)
From the Bottom Up
400(2)
From the Middle Out
402(8)
Perspective
410(9)
References
410(9)
Polymeric Monolithic Capillary Columns in Proteomics
419(20)
Alexander R. Ivanov
Introduction and Overview
419(2)
High-Efficiency Peptide Analysis on Monolithic Multimode Capillary Columns
421(4)
High-Sensitivity ESI-MS Analysis of Protein Tryptic Digests Using Ultra Low I.D. Polystyrene-Divinylbenzene Monolithic Capillary Columns
425(4)
Polystyrene Monolithic Capillary Columns in On-Line 2D LC-ESI-MS Proteomic Analysis
429(2)
Conclusion
431(8)
Acknowledgments
431(1)
References
431(8)
Part V Related Techniques
Proteins Staining in Polyacrylamide Gels
439(14)
Gary B. Smejkal
Introduction
439(1)
Organic Dyes
440(2)
Staining Proteins with Tannins
442(1)
Counterion Stains
442(2)
Silver Staining
444(1)
Negative Stains
445(1)
Schlieren Optics
445(1)
Fluorescent Stains
446(2)
Recent Developments in Colloidal Coomassie Chemistry
448(5)
Acknowledgments
450(1)
References
450(3)
Multiplexed Proteomics: Fluorescent Detection of Proteins, Glycoproteins, and Phosphoproteins in Two-Dimensional (2D) Gels
453(8)
Birte Aggeler-Schulenberg
Introduction
453(1)
Materials
454(3)
2D Gel Staining
454(1)
Glycoprotein Detection Using Pro-Q Emerald 300 Glycoprotein Gel Stain
455(1)
Phosphoprotein Detection in 2D Gels
455(1)
Total Protein Detection Using SYPRO Ruby Protein Gel Stain
456(1)
Instrumentation for Imaging Fluorescent Stains and Data Analysis
456(1)
Methods
457(1)
Detection of Phosphoproteins
457(1)
Detection of Glycoproteins
458(1)
Detection of Total Protein
458(1)
Three-Color Staining of Gels
458(1)
Conclusions
458(3)
References
460(1)
Glyoxyl Agarose and Its Composite Gels: Advantageous Alternatives to Polyacrylamide Gel Electrophoresis for Very Large and Small Proteins and Peptides and All Sizes in Between
461(6)
John R. Shainoff
Introduction
461(2)
Preparation of Glyoxyl Agarose
463(1)
Composite Gels
463(1)
Simplified Immunoprobing with Compressed Gels
463(1)
Mass Production of the Composites
464(1)
Current Availability
464(1)
Conclusion
464(3)
References
464(3)
Nuclear Magnetic Resonance--Driven Chemical Proteomics: The Functional and Mechanistic Complement to Proteomics
467(22)
Phani Kumar Pullela
Daniel S. Sem
Introduction
468(1)
Background
468(5)
Chemical Proteomics as Systems-Based Characterization of Protein Function
468(1)
NMR-Based Characterization of Protein Structure
469(2)
Protein Dynamics: From Snapshots to Movies
471(1)
NMR-Based Fragment Assembly: Modular Inhibitor Design in NMR Legoland™
471(1)
The Emerging Role of NMR Spectroscopy in Chemical Proteomics
472(1)
Chemical Proteomic Applications of NMR In Vitro
473(7)
A NMR-Guided Design of Gene-Family-Focused Libraries of Chemical Proteomic Probes Using NMR Solve
473(3)
Cofactor Fingerprinting with STD NMR: Addressing the Needs of Functional Proteomics by Identifying Binding Preferences
476(2)
Ligand Docking Using NMR Constraints: T1 Relaxation or NOE Data
478(2)
Chemical Proteomic Applications of NMR In Vivo
480(4)
In Cell NMR: Chemical Proteomics in the Cellular Milieu
480(1)
Molecular Imaging: Magnetic Resonance Contrast Agents as Chemical Proteomic Probes in Whole Organisms
481(3)
Summary
484(5)
References
484(5)
Electrophoretic Nuclear Magnetic Resonance in Proteomics: Toward High-Throughput Structural Characterization of Biological Signaling Processes
489(16)
Qiuhong He
Xiangjin Song
Introduction
489(2)
Separating Protein Signals without Physical Separation
491(3)
Structural Characterization of Coexisting Proteins in Solution
494(1)
Heat-Induced Convection in Biological Buffer Solutions
495(4)
High-Throughput Structural Determination of Protein Active Pockets
499(2)
Conclusions
501(4)
Acknowledgments
502(1)
References
502(3)
Index 505


Gary B. Smejkal, Alexander Lazarev