Protein and Peptide Analysis by LC-MS

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ISBN: 9781849731829 | 1849731829
Cover: Hardcover
Copyright: 8/31/2011

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This book is the first example in presenting LC-MS strategies for the analysis of peptides and proteins with detailed information and hints about the needs and problems described from experts on-the-job. The best advantage is -for sure- the practical insight of experienced analysts into their novel protein analysis techniques. Readers starting in 'Proteomics' should be able to repeat each experiment with own equipment and own protein samples, like clean-up, direct protein analysis, after (online) digest, with modifications and others. Furthermore, the reader will learn more about strategies in protein analysis, like quantitative analysis, industrial standards, functional analysis and more.

Contributors	p. xix
Top Down and Bottom Up Analysis of Proteins (Focusing on Quantitative Aspects)	p. 1
Introduction	p. 1
Quantitative Proteomics	p. 3
Quantitative Proteomics by Label-Free Techniques	p. 3
Quantitative Proteomics by Isotopic Labelling Techniques	p. 4
Isobaric Tags for Relative and Absolute Quantification	p. 7
Absolute Quantification in Proteomics with Targeted Proteomics	p. 7
Absolute Quantification Using SRM	p. 8
Notes	p. 9
References	p. 9
How to Couple and Handle Liquid Chromatography with Mass Spectrometry	p. 11
Introduction	p. 11
Separation	p. 11
Ionization	p. 12
Mass Spectrometric Detection	p. 13
Materials and System	p. 15
Chemicals	p. 15
HPLC and Mass Spectrometer	p. 15
Methods	p. 16
Molecular Conditions	p. 18
Spray Conditions (ESI)	p. 21
Spray Conditions (APCI)	p. 22
Spray Conditions (MMI; i.e. ESI + APCI)	p. 23
Notes	p. 23
References	p. 24
Expression and Purification of Bioactive Proteins/Peptides with Conventional Liquid Chromatography	p. 26
Introduction	p. 26
Experimental	p. 30
Materials	p. 30
FPLC System	p. 30
Cloning cDNA and Construction of the Expression Vector	p. 30
Recombinant Protein Expression in E. coli	p. 30
Separation by Ion Exchange Chromatography	p. 31
Separation by Gel Filtration Chromatography	p. 31
Separation by Hydrophobic Interaction Chromatography	p. 31
Results and Discussion	p. 31
Ion Exchange Chromatography	p. 31
Gel Filtration Chromatography	p. 32
Hydrophobic Interaction Chromatography	p. 33
Overall Evaluation	p. 34
Notes	p. 34
Other Hosts for Expression	p. 34
Ion Exchange Chromatography	p. 34
Gel Filtration Chromatography	p. 35
Hydrophobic Interaction Chromatography	p. 36
References	p. 36
Liquid Chromatography-Mass Spectrometry of Intact Proteins	p. 38
Introduction	p. 38
Liquid Chromatography	p. 39
Understanding Proteins	p. 41
HPLC Instrumentation	p. 42
Stationary Phase Morphology	p. 43
Column Temperature	p. 43
Mobile Phase Composition	p. 44
Matrix Effects	p. 44
Sample Preparation	p. 45
Choice of Stationary-Phase Chemistry	p. 47
Two-Dimensional Liquid Chromatography	p. 50
Mass Spectrometry	p. 51
LC-MS Profiling/Quantification	p. 51
Conformational Analysis and Protein-Protein Interactions	p. 52
Top Down Sequencing	p. 52
Notes	p. 55
References	p. 55
LC-MS(/MS) of Trypsin-Hydrolysed Proteins	p. 56
Introduction	p. 56
Hydrolysis Materials and Equipment	p. 61
Enzymatic Hydrolysis of ß-Lactoglobulin	p. 61
Tips with Enzymes	p. 66
LC-ESI-TOF/MS Spectra Equipment and Methods	p. 66
Equipment	p. 66
Peptide Mass Fingerprinting	p. 67
Data Analysis	p. 67
Tips with MS Data from Trypsin Hydrolysates	p. 67
Conclusions	p. 68
References	p. 68
On-line Protein Digestion in Combination with Chromatographic Separation and Mass Spectrometric Detection	p. 71
Introduction	p. 71
Proteolysis of Proteins	p. 72
Immobilized Enzyme Reactors	p. 74
Methods Employing IMERs in Hyphenated Analytical Systems	p. 76
Methods Employing In-Solution Digestion in Continuous-Flow Reactors	p. 80
Notes and Hints	p. 83
References	p. 85
Bioinformatic Tools for the LC-MS/MS Analysis of Proteins and Peptides	p. 87
Introduction	p. 87
General	p. 87
Protein and Peptide Sequence Analysis by MS/MS	p. 88
Software Tools for Peptide Sequence Interpretation by MS/MS	p. 88
Quantification by LC-MS/MS after Isotopic Labelling	p. 89
Materials	p. 90
Peptides	p. 90
LC-MS	p. 90
Protein Identification by MS/MS	p. 91
Protein Quantification	p. 91
Data Analysis and Interpretation	p. 91
Methods	p. 91
Sample Preparation for LC-MS	p. 91
LC-MS Analysis	p. 92
Exemplary MS/MS Data Analysis by Database Comparison	p. 92
Automated MS/MS Data Analysis for Quantification	p. 95
Identification of Significantly Different Proteins	p. 98
Data Interpretation	p. 100
Notes	p. 100
MS/MS Database Searches	p. 100
Quantitative Determinations by LC-MS/MS	p. 101
References	p. 102
Quantitative LC-MS of Proteins	p. 104
Introduction	p. 104
Materials	p. 105
SILAC Labelling	p. 105
Sample Preparation	p. 106
LC-MS Analysis	p. 106
Equipment	p. 106
Methods	p. 106
Preparation of SILAC Medium	p. 107
SILAC Labelling and Incorporation Test	p. 108
SILAC Experiment	p. 109
Data Analysis	p. 111
Labelling Check	p. 111
SILAC Experiment	p. 115
Notes	p. 119
References	p. 120
LC-MS for the Identification of Post-Translational Modifications of Proteins	p. 123
Introduction	p. 123
Materials	p. 125
In-Solution Protein Digestion	p. 125
Strong Cation Exchange (SCX) Chromatography	p. 125
Titanium Oxide (TiO₂) Chromatography	p. 125
Liquid Chromatography - Mass Spectrometry (LC-MS)	p. 125
Methods	p. 126
In-Solution Protein Digestion	p. 126
SCX Chromatography	p. 126
TiO₂ Chromatography	p. 127
LC-MS	p. 128
References	p. 131
LC-MS for the Determination of the Enzymatic Activity of Proteins	p. 133
Introduction	p. 133
Materials	p. 134
The Example Assay	p. 134
Chemicals	p. 134
Mass Spectrometer	p. 134
LC-MS Method	p. 134
Direct Infusion	p. 135
Robot Infusion	p. 135
Methods	p. 135
LC-MS	p. 135
Direct Infusion or Robot Infusion Measurement	p. 136
Data Analysis	p. 138
Comparison of LC-MS, Direct Infusion and Robot Infusion	p. 139
Notes	p. 139
General Remarks for Working with Enzymes	p. 139
General Remarks for Mass Spectrometric Applications	p. 140
Remarks Regarding MS-Based Enzymatic Assays	p. 141
References	p. 141
Functional Analysis of Proteins, Including LC-MS and Special Freeware	p. 142
Introduction	p. 142
Materials and Systems	p. 143
Chemicals	p. 143
The Example Enzymatic Assay	p. 143
HPLC (Sample Introduction)	p. 143
Mass Spectrometer	p. 143
Complex Study with Flow Injection Analysis (FIA)	p. 144
Analysis Software	p. 144
Methods	p. 144
Studying Complex Formation (and Simultaneous Reaction Inhibition) with Continuous-Flow Mixing Analysis	p. 146
Data Analysis	p. 148
Notes	p. 154
Working with Enzymes and MS-Based Enzymatic Assays	p. 154
Working with Achroma External Analytical Software	p. 154
References	p. 155
Industrial Standards and Strategies in LC-MS Analysis of Proteins	p. 156
Introduction	p. 156
Materials and Instruments	p. 157
Reagents, Solvents and Chemicals	p. 157
Analytical Columns and Desalting Cartridges	p. 157
HPLC	p. 157
Preparative Liquid Chromatography	p. 158
Mass Spectrometer	p. 158
Direct Infusion	p. 158
Methods	p. 158
Mass Spectrometric Detection	p. 158
Protein Mass Spectra Deconvolution	p. 158
Intact Protein Analysis by Direct Infusion ESI-MS	p. 159
HPLC-ESI-MS	p. 160
Introducing Selectivity for ESI-MS Protein Analysis by Chemical Modification	p. 162
References	p. 166
Subject Index	p. 168
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Protein and Peptide Analysis by LC-MS

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