Protein and Peptide Analysis by LC-MS -- Contents -- Contributors -- CHAPTER 1 Top Down and Bottom Up Analysis of Proteins (Focusing on Quantitative Aspects) -- 1.1 Introduction -- 1.2 Quantitative Proteomics -- 1.2.1 Quantitative Proteomics by Label-Free Techniques -- 1.2.2 Quantitative Proteomics by Isotopic Labelling Techniques -- 1.2.3 Isobaric Tags for Relative and Absolute Quantification -- 1.2.4 Absolute Quantification in Proteomics with Targeted Proteomics -- 1.2.5 Absolute Quantification Using SRM -- 1.3 Notes -- References -- CHAPTER 2 How to Couple and Handle Liquid Chromatography with Mass Spectrometry -- 2.1 Introduction -- 2.1.1 Separation -- 2.1.2 Ionization -- 2.1.3 Mass Spectrometric Detection -- 2.2 Materials and System -- 2.2.1 Chemicals -- 2.2.2 HPLC and Mass Spectrometer -- 2.3 Methods -- 2.3.1 Molecular Conditions -- 2.3.2 Spray Conditions (ESI) -- 2.3.3 Spray Conditions (APCI) -- 2.3.4 Spray Conditions (MMI -- i.e. ESI+APCI) -- 2.4 Notes -- References -- CHAPTER 3 Expression and Purification of Bioactive Proteins/Peptides with Conventional Liquid Chromatography -- 3.1 Introduction -- 3.2 Experimental -- 3.2.1 Materials -- 3.2.2 FPLC System -- 3.2.3 Cloning cDNA and Construction of the Expression Vector -- 3.2.4 Recombinant Protein Expression in E. coli -- 3.2.5 Separation by Ion Exchange Chromatography -- 3.2.6 Separation by Gel Filtration Chromatography -- 3.2.7 Separation by Hydrophobic Interaction Chromatography -- 3.3 Results and Discussion -- 3.3.1 Ion Exchange Chromatography -- 3.3.2 Gel Filtration Chromatography -- 3.3.3 Hydrophobic Interaction Chromatography -- 3.3.4 Overall Evaluation -- 3.4 Notes -- 3.4.1 Other Hosts for Expression -- 3.4.2 Ion Exchange Chromatography -- 3.4.3 Gel Filtration Chromatography -- 3.4.4 Hydrophobic Interaction Chromatography -- References.

CHAPTER 4 Liquid Chromatography-Mass Spectrometry of Intact Proteins -- 4.1 Introduction -- 4.2 Liquid Chromatography -- 4.2.1 Understanding Proteins -- 4.2.2 HPLC Instrumentation -- 4.2.3 Stationary Phase Morphology -- 4.2.4 Column Temperature -- 4.2.5 Mobile Phase Composition -- 4.2.6 Matrix Effects -- 4.2.7 Sample Preparation -- 4.2.8 Choice of Stationary-Phase Chemistry -- 4.2.9 Two-Dimensional Liquid Chromatography -- 4.3 Mass Spectrometry -- 4.3.1 LC-MS Profiling/Quantification -- 4.3.2 Conformational Analysis and Protein-Protein Interactions -- 4.3.3 Top Down Sequencing -- 4.4 Notes -- References -- CHAPTER 5 LC-MS(/MS) of Trypsin-Hydrolysed Proteins -- 5.1 Introduction -- 5.2 Hydrolysis Materials and Equipment -- 5.2.1 Enzymatic Hydrolysis of β-Lactoglobulin -- 5.2.2 Tips with Enzymes -- 5.3 LC-ESI-TOF/MS Spectra Equipment and Methods -- 5.3.1 Equipment -- 5.3.2 Peptide Mass Fingerprinting -- 5.4 Data Analysis -- 5.4.1 Tips with MS Data from Trypsin Hydrolysates -- 5.5 Conclusions -- References -- CHAPTER 6 On-line Protein Digestion in Combination with Chromatographic Separation and Mass Spectrometric Detection -- 6.1 Introduction -- 6.2 Proteolysis of Proteins -- 6.3 Immobilized Enzyme Reactors -- 6.4 Methods Employing IMERs in Hyphenated Analytical Systems -- 6.5 Methods Employing In-Solution Digestion in Continuous-Flow Reactors -- 6.6 Notes and Hints -- References -- CHAPTER 7 Bioinformatic Tools for the LC-MS/MS Analysis of Proteins and Peptides -- 7.1 Introduction -- 7.1.1 General -- 7.1.2 Protein and Peptide Sequence Analysis by MS/MS -- 7.1.3 Software Tools for Peptide Sequence Interpretation by MS/MS -- 7.1.4 Quantification by LC-MS/MS after Isotopic Labelling -- 7.2 Materials -- 7.2.1 Peptides -- 7.2.2 LC-MS -- 7.2.3 Protein Identification by MS/MS -- 7.2.4 Protein Quantification -- 7.2.5 Data Analysis and Interpretation.

7.3 Methods -- 7.3.1 Sample Preparation for LC-MS -- 7.3.2 LC-MS Analysis -- 7.3.3 Exemplary MS/MS Data Analysis by Database Comparison -- 7.3.4 Automated MS/MS Data Analysis for Quantification -- 7.3.5 Identification of Significantly Different Proteins -- 7.3.6 Data Interpretation -- 7.4 Notes -- 7.4.1 MS/MS Database Searches -- 7.4.2 Quantitative Determinations by LC-MS/MS -- References -- CHAPTER 8 Quantitative LC-MS of Proteins -- 8.1 Introduction -- 8.2 Materials -- 8.2.1 SILAC Labelling -- 8.2.2 Sample Preparation -- 8.2.3 LC-MS Analysis -- 8.2.4 Equipment -- 8.3 Methods -- 8.3.1 Preparation of SILAC Medium -- 8.3.2 SILAC Labelling and Incorporation Test -- 8.3.3 SILAC Experiment -- 8.4 Data Analysis -- 8.4.1 Labelling Check -- 8.4.2 SILAC Experiment -- 8.5 Notes -- References -- CHAPTER 9 LC-MS for the Identification of Post-Translational Modifications of Proteins -- 9.1 Introduction -- 9.2 Materials -- 9.2.1 In-Solution Protein Digestion -- 9.2.2 Strong Cation Exchange (SCX) Chromatography -- 9.2.3 Titanium Oxide (TiO2) Chromatography -- 9.2.4 Liquid Chromatography - Mass Spectrometry (LC-MS) -- 9.3 Methods -- 9.3.1 In-Solution Protein Digestion -- 9.3.2 SCX Chromatography -- 9.3.3 TiO2 Chromatography -- 9.3.4 LC-MS -- References -- CHAPTER 10 LC-MS for the Determination of the Enzymatic Activity of Proteins -- 10.1 Introduction -- 10.2 Materials -- 10.2.1 The Example Assay -- 10.2.2 Chemicals -- 10.2.3 Mass Spectrometer -- 10.2.4 LC-MS Method -- 10.2.5 Direct Infusion -- 10.2.6 Robot Infusion -- 10.3 Methods -- 10.3.1 LC-MS -- 10.3.2 Direct Infusion or Robot Infusion Measurement -- 10.3.3 Data Analysis -- 10.3.4 Comparison of LC-MS, Direct Infusion and Robot Infusion -- 10.4 Notes -- 10.4.1 General Remarks for Working with Enzymes -- 10.4.2 General Remarks for Mass Spectrometric Applications.

10.4.3 Remarks Regarding MS-Based Enzymatic Assays -- References -- CHAPTER 11 Functional Analysis of Proteins, Including LC-MS and Special Freeware -- 11.1 Introduction -- 11.2 Materials and Systems -- 11.2.1 Chemicals -- 11.2.2 The Example Enzymatic Assay -- 11.2.3 HPLC (Sample Introduction) -- 11.2.4 Mass Spectrometer -- 11.2.5 Complex Study with Flow Injection Analysis (FIA) -- 11.2.6 Analysis Software -- 11.3 Methods -- 11.3.1 Studying Complex Formation (and Simultaneous Reaction Inhibition) with Continuous-Flow Mixing Analysis -- 11.3.2 Data Analysis -- 11.4 Notes -- 11.4.1 Working with Enzymes and MS-Based Enzymatic Assays -- 11.4.2 Working with Achroma External Analytical Software -- References -- CHAPTER 12 Industrial Standards and Strategies in LC-MS Analysis of Proteins -- 12.1 Introduction -- 12.2 Materials and Instruments -- 12.2.1 Reagents, Solvents and Chemicals -- 12.2.2 Analytical Columns and Desalting Cartridges -- 12.2.3 HPLC -- 12.2.4 Preparative Liquid Chromatography -- 12.2.5 Mass Spectrometer -- 12.2.6 Direct Infusion -- 12.3 Methods -- 12.3.1 Mass Spectrometric Detection -- 12.3.2 Protein Mass Spectra Deconvolution -- 12.3.3 Intact Protein Analysis by Direct Infusion ESI-MS -- 12.3.4 HPLC-ESI-MS -- 12.3.5 Introducing Selectivity for ESI-MS Protein Analysis by Chemical Modification -- References -- Subject Index.