Protein Sequencing and Identification Using Tandem Mass Spectrometry

by Kinter, Michael; Sherman, Nicholas E.

• ISBN13: 9780471322498
• ISBN10: 0471322490
• Edition: 1st
• Format: Hardcover
• Copyright: 2000-10-02
• Publisher: Wiley-Interscience

Summary

How to design, execute, and interpret experiments for protein sequencing using mass spectrometry The rapid expansion of searchable protein and DNA databases in recent years has triggered an explosive growth in the application of mass spectrometry to protein sequencing. This timely and authoritative book provides professionals and scientists in biotechnology research with complete coverage of procedures for analyzing protein sequences by mass spectrometry, including step-by-step guidelines for sample preparation, analysis, and data interpretation. Michael Kinter and Nicholas Sherman present their own high-quality, laboratory-tested protocols for the analysis of a wide variety of samples, demonstrating how to carry out specific experiments and obtain fast, reliable results with a 99uccess rate. Readers will get sufficient experimental detail to apply in their own laboratories, learn about the proper selection and operation of instruments, and gain essential insight into the fundamental principles of mass spectrometry and protein sequencing. Coverage includes: Peptide fragmentation and interpretation of product ion spectra Basic polyacrylamide gel electrophoresis Preparation of protein digests for sequencing experiments Mass spectrometric analysis using capillary liquid chromatography Techniques for protein identification by database searches Characterization of modified peptides using tandem mass spectrometry And much more

Author Biography

MICHAEL KINTER, PhD, is an associate staff member in the Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation.

Table of Contents

Series Preface	xiii
Preface	xv
An Introduction to Protein Sequencing Using Tandem Mass Spectrometry	1	(5)
Introduction	1	(4)
References	5	(1)
The Primary Structure of Proteins and a Historical Overview of Protein Sequencing	6	(23)
Protein and Peptide Structure	6	(4)
Edman Degradation	10	(5)
The Edman Reaction	10	(1)
Incorporation of the Edman Degradation Reaction into Automated Protein Sequenators	11	(3)
Edman Degradation in Proteomic Research	14	(1)
Tandem Mass Spectrometry	15	(7)
A Brief History of the Application of Mass Spectrometry to Protein Sequencing	15	(1)
Sequence Analysis of Peptides Using Electron Ionization Mass Spectrometry	16	(1)
The Utilization of Fast Atom Bombardment with Tandem Mass Spectrometry to Sequence Peptides	17	(2)
Internal Sequence Analysis of Proteins Using Electrospray Ionization-Tandem Mass Spectrometry and Matrix-Assisted Laser Desorption/Ionization-Time-of-Flight Mass Spectrometry	19	(3)
Summary	22	(1)
References	23	(6)
Fundamental Mass Spectrometry	29	(35)
An Overview of the Instrumentation	29	(2)
Ionization Methods	31	(8)
Electrospray Ionization	32	(4)
Nanospray and Microspray Ionization	36	(1)
Matrix-Assisted Laser Desorption/ Ionization	37	(2)
Mass Analyzers	39	(13)
Fundamental Parameters of Mass Analysis	40	(3)
Quadrupole Mass Filters (3.20)	43	(2)
Ion Trap Mass Analyzers (3.21--3.23)	45	(3)
Time-of-Flight Mass Analyzers (3.5)	48	(4)
Tandem Mass Spectrometry	52	(7)
Collisionally Induced Dissociation	52	(1)
Tandem Mass Spectrometers	53	(4)
Types of Tandem Mass Spectrometry Experiments	57	(2)
Data Systems	59	(2)
Summary	61	(1)
References	61	(3)
Collisionally Induced Dissociation of Protonated Peptide Ions and the Interpretation of Product Ion Spectra	64	(53)
Introduction	64	(1)
Peptide Fragmentation Chemistry	65	(16)
Collisionally Induced Dissociation of Peptide Ions Formed by Electrospray Ionization	66	(13)
Fragmentation of Protonated Peptide Ions Formed by Matrix-Assisted Laser Desorption/Ionization	79	(2)
Interpretation of the Product Ion Spectra of Tryptic Peptides	81	(33)
Tabulated Values Used in the Interpretation	81	(5)
A Strategy for the Interpretation of Product Ion Spectra of Tryptic Peptides	86	(3)
Sample Interpretation Problem Number One	89	(5)
Sample Interpretation Problem Number Two	94	(5)
A Summary of Interpretation Problems One and Two	99	(1)
Examples of More Difficult Product Ion Spectra That Cannot Be Completely Interpreted	100	(10)
Interpretation of Product Ion Spectra from Triply Charged Ions	110	(4)
Summary	114	(1)
References	115	(2)
Basic Polyacrylamide Gel Electrophoresis	117	(30)
Introduction	117	(1)
The Principles of Gel Electrophoresis	118	(6)
Protein Movement and Separation	118	(5)
Protein Detection	123	(1)
The Basic Steps in a Polyacrylamide Gel Electrophoresis Experiment	124	(9)
SDS-PAGE Gels for Protein Molecular Weight Measurements	125	(3)
Isoelectric Focusing Gels	128	(3)
Protein Detection	131	(2)
Data Analysis	133	(1)
The Protein Sample Presented for Digestion and Amino Acid Sequence Analysis	133	(2)
Example Protocols for 2D Electrophoresis with Immobilized pH-Gradient Gels	135	(10)
A Protocol for the Preparation of a Protein Homogenate, from Cultured Mammalian Cells, for Isoelectric Focusing in an Immobilized pH-Gradient Gel	135	(3)
A Protocol for the Passive Rehydration of Immobilized pH-Gradient Gels	138	(1)
A Protocol for the Active Rehydration of Immobilized pH-Gradient Gels	139	(1)
A Protocol for the Equilibration, with Reduction and Alkylation, of an Immobilized pH-Gradient Gel for Molecular Weight Analysis by SDS-PAGE	140	(1)
A Protocol for Staining Polyacrylamide Gels with Coomassie Blue	141	(2)
A Protocol for Staining Polyacrylamide Gels with Colloidal Coomassie Blue	143	(1)
A Protocol for Silver-Staining Polyacrylamide Gels	144	(1)
Summary	145	(1)
References	146	(1)
The Preparation of Protein Digests for Mass Spectrometric Sequencing Experiments	147	(19)
Introduction	147	(1)
Protease Selection	148	(2)
The Effects of Contamination	150	(2)
An In-Gel Digestion Protocol	152	(8)
Protein Band Selection	153	(1)
The In-Gel Digestion Protocol	153	(7)
An In-Solution Digestion Protocol	160	(3)
The In-Solution Digestion Protocol	161	(2)
Summary	163	(1)
References	164	(2)
Mass Spectrometric Analysis of Tryptic Digests	166	(41)
Introduction	166	(2)
Mass Spectrometric Analysis of Tryptic Digests Using Capillary Column Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry	168	(26)
Capillary Column Liquid Chromatography	168	(4)
The Acquisition and Presentation of Mass Spectra	172	(2)
Chromatogram Reconstruction	174	(2)
The Determination of Peptide Molecular Weights	176	(2)
The Characterization of Peptide Structure	178	(1)
Automated Acquisition of Both Mass Spectra and Product Ion Spectra	179	(3)
Benchmark Performance of a Capillary Column Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry Experiment	182	(8)
Improving Sensitivity Through the Use of Microspray Ionization	190	(1)
A Protocol for Packing a Capillary Liquid Chromatography Column	191	(3)
Mass Spectrometric Analysis of Tryptic Digests by Matrix-Assisted Laser Desorption/Ionization-Time-of-Flight Mass Spectrometry	194	(12)
The Acquisition and Presentation of Mass Spectra	195	(2)
The Determination of Peptide Molecular Weights	197	(2)
The Characterization of Peptide Structure	199	(1)
Automated Data Acquisition	200	(1)
Benchmark Performance of a Delayed Extraction-Reflectron Time-of-Flight Mass Spectrometry Experiment	201	(2)
A Protocol for Sample Desalting by Solid-Phase Extraction Prior to Analysis	203	(3)
Summary	206	(1)
Reference	206	(1)
Protein Identification by Database Searching	207	(31)
Introduction	207	(1)
The Sequence Databases	208	(4)
The Protein Sequence Databases	208	(3)
The Genomic and Expressed Sequence Tag Databases	211	(1)
Database Search Programs for Use with Mass Spectrometric Protein Sequencing Data	212	(22)
Database Searching with Amino Acid Sequences	212	(7)
Database Searching with Peptide Molecular Weights	219	(7)
Database Searching with Uninterpreted Product Ion Spectra	226	(8)
Reporting the Results of a Protein Identification	234	(1)
Summary	235	(1)
References	236	(2)
Sequence Analysis of Novel Proteins	238	(31)
Introduction	238	(3)
The Role of Confirmation in the Interpretation Process	241	(1)
Strategies to Enhance and Confirm the Interpretation of Product Spectra	241	(24)
Fragmentation of Product Ions	243	(8)
Synthetic Peptides	251	(3)
Peptide Derivatization	254	(5)
Edman Degradation	259	(6)
Using Mass Spectrometric Data to Verify the Results of Cloning Experiments	265	(2)
Summary	267	(1)
References	267	(2)
The Characterization of Post-Translationally Modified Proteins Using Tandem Mass Spectrometry	269	(26)
Introduction	269	(3)
An Overview of the Methods	272	(7)
Identifying Modified Peptides Ions by Searching for Calculated Molecular Weights	273	(3)
Identifying Modified Peptides by Observation of Specific Fragmentation Reactions	276	(1)
Isolating Modified Peptides by Affinity Chromatography Prior to Tandem Mass Spectrometric Characterization	277	(2)
Examples of Experiments Characterizing Specific Post-Translational Modifications	279	(11)
Phosphorylation	280	(5)
Xenobiotic Modification	285	(2)
Carbohydrate Modification	287	(3)
Summary	290	(1)
References	290	(5)
Index	295