Note: Microcharacterization of Proteins -- Contents -- Section I: Overview -- I.1 Microcharacterization of Proteins -- 1 General Aspects -- 2 From a Cell to a Protein Sequence -- 3 Genome and Proteome -- Section II: Microseparation Techniques and Sample Preparation -- II.1 Microseparation Techniques I: High Resolution Gel-Electrophoretic Techniques : Qualitative, Quantitative and Micropreparative Applications -- 1 Introduction -- 2 Theory -- 3 Media and Equipment -- 3.1 Media -- 3.2 Equipment -- 4 Gel Electrophoretic Methods -- 4.1 Disc Electrophoresis -- 4.2 Gradient Gel Electrophoresis -- 4.3 Additives in Electrophoresis -- 4.4 SDS Electrophoresis -- 4.5 Isoelectric Focusing -- 4.6 Two-Dimensional Electrophoresis (2 -DE) -- 5 References -- II.2 Microseparation Techniques II: Gel Electrophoresis for Sample Preparation in Protein Chemistry -- 1 Introduction -- 2 Denaturing Techniques -- 2.1 Commonly Used SDS-Polyacrylamide Gel Electrophoresis Techniques for Protein Separation -- 2.2 Blue-SDS-PAGE for Recovery of Membrane Proteins from Gels -- 2.3 Electroelution of Proteins after Blue-SDS-PAGE -- 2.4 Electroblotting of Blue and Colorless SDS Gels -- 2.5 Isoelectric Focusing in the Presence of Urea -- 3 Native Techniques -- 3.1 Colorless-Native-PAGE -- 3.2 Blue-Native-PAGE -- 3.3 Native Isoelectric Focusing -- 4 References -- II.3 Microseparation Techniques III: Electroblotting -- 1 Introduction -- 2 Electroblotting -- 2.1 Polyacrylamide Gel Electrophoresis -- 2.2 Blot Systems -- 2.2.1 Tank Blotting -- 2.2.2 Semidiy Blotting -- 2.3 Blotting Parameters -- 2.3.1 The Blotting Process -- 2.3.2 Transfer Buffers -- 2.3.3 Addition of SDS -- 2.3.4 Addition of Methanol -- 2.3.5 Influence of Protein Concentration -- 3 Blotting Membranes -- 4 References -- II.4 Microseparation Techniques IV: Analysis of Peptides and Proteins by Capillary Electrophoresis. , 1 Introduction -- 2 Theory -- 2.1 Capillary Isotachophoresis -- 2.2 Capillary Zone Electrophoresis -- 2.3 Electroosmotic Flow -- 3 Instrumentation -- 3.1 Injection -- 3.2 Detection -- 4 Applications -- 4.1 Peptide Separations -- 4.2 Protein Separations -- 5 References -- II.5 Microseparation Techniques V: High Performance Liquid Chromatography -- 1 Introduction -- 2 Principle of HPLC -- 3 Getting Started -- 3.1 Solvents -- 3.2 Pump -- 3.3 Gradient -- 3.4 Pre-Column Split -- 3.5 Sample Preparation -- 3.6 Injector -- 3.7 Tubings -- 3.8 In-Line Filter, Guard Column -- 3.9 Column -- 3.10 Detection -- 3.11 Fractionation -- 4 Applications -- 5 References -- II.6 Sample Preparation I: Removal of Salts and Detergents -- 1 Introduction -- 2 Removal of Salts or Polar Components -- 2.1 Protein-Binding Membranes -- 2.2 High Performance Liquid Chromatography -- 2.3 Desalting on Microcolumns -- 3 Removal of Detergents and Apolar Contaminants -- 3.1 Detergents -- 3.2 Protein-binding Membranes -- 3.3 Precipitation -- 3.4 SDS-PAGE -- 3.5 Concentrating Gels -- 4 "Golden Rules" for Protein and Peptide Handling -- 5 References -- II.7 Sample Preparation II: Chemical and Enzymatic Fragmentation of Proteins -- 1 Strategy -- 2 Denaturation, Reduction and Alkylation -- 3 Enzymatic Fragmentation -- 3.1 Enzymes -- 3.2 Practical Considerations -- 3.2.1 Practical Considerations for On-Membrane Digestions -- 3.2.2 Practical Considerations for In-Gel Digestions -- 3.2.3 Automation of Digest Procedures -- 4 Chemical Fragmentation -- 4.1 Cyanogen Bromide Cleavage -- 4.2 Partial Acid Hydrolysis -- 4.3 Hydroxylamine Cleavage of Asn-Gly Bonds -- 4.4 Cleavage at Tryptophan -- 4.5 Cleavage at Cysteine -- 5 References -- Section III: Bioanalytical Characterization -- III.1 Amino Acid Analysis -- 1 Introduction -- 2 Sample Preparation -- 2.1 Peptides and Proteins. , 2.1.1 Enzymatic Hydrolysis -- 2.1.2 Acid Hydrolysis -- 2.1.3 Alkaline Hydrolysis -- 2.2 Free Amino Acids -- 3 Derivatization -- 3.1 Post-Column Derivatization -- 3.1.1 Ninhydrin -- 3.1.2 Orthophthaldialdehyde -- 3.1.3 Fluorescamine -- 3.2 Pre-Column Derivatization -- 3.2.1 Phenylisothiocyanate -- 3.2.2 Orthophthaldialdehyde -- 3.2.3 Fluorenylmethyl Chloroformate -- 3.2.4 Dabsyl Chloride -- 3.2.5 Dansyl Chloride -- 3.2.6 Chiral Reagents -- 4 Data Evaluation -- 5 Instrumentation -- 6 Discussion -- 7 References -- III.2 Chemical Methods for Protein Sequence Analysis -- 1 The Edman Degradation -- 1.1 Coupling, Cleavage and Conversion -- 1.2 Identification of the PTH Amino Acids -- 2 Instrumentation -- 2.1 The Liquid Phase Sequencer -- 2.2 The Solid Phase Sequencer -- 2.3 The Gas Phase Sequencer -- 2.4 The Pulsed Liquid Phase Sequencer -- 2.5 The Biphasic Column Sequencer -- 3 Difficulties of Amino Acid Sequence Analysis -- 3.1 The Sample and Sample Matrices -- 3.2 Difficulties with the Edman Chemistry -- 4 C-Terminal Sequence Analysis -- 4.1 Chemical Degradation -- 4.2 Enzymatic Methods -- 4.3 Methods Combined with Mass Spectrometry -- 5 References -- III.3 Analyzing Post-Translational Protein Modifications -- 1 Introduction -- 2 Classification of Post-Translational Modifications According to their Behavior during Purification and Edman Degradation -- 2.1 Modifications: Stable during Purification and Edman Degradation -- 2.2 Modifications : Stable during Purification but Unstable during Edman Degradation -- 2.3 Modifications : Unstable during Purification and Edman Degradation -- 3 Examples -- 3.1 1-Methyl-Histidine -- 3.2 Glyco-Asparagine, Glyco-Threonine -- 3.3 Phospho-Tyrosine -- 3.4 N-Pyruvyl or N-a-Oxo-Butyric Acid -- 3.5 Gluco-Arginine -- 3.6 Farnesyl-Cysteine -- 3.7 Phospho-Serine -- 3.8 Phospho-Threonine. , 3.9 Screening for Phospho-Serine/Threonine Containing Peptides by HPLC/MS -- 3.10 Lanthionine, 3 -Methyl-Lanthionine, Dehydroalanine, Dehydro-a-aminobutyric Acid -- 4 References -- III.4 Analysis of Biopolymers by Matrix-Assisted Laser Desorption/Ionization (MALDI) Mass Spectrometry * -- 1 Introduction -- 2 Development of MALDI -- 2.1 Mechanism of Matrix-Assisted Laser Desorption/Ionization -- 3 Instrumentation -- 3.1 Time-of-Flight (TOF) Mass Spectrometers -- 3.2 Laser Desorption Ion Source -- 3.3 Ion Detection and Data Collection -- 4 Applications -- 4.1 Sample Preparation -- 4.2 Ion Fragmentation -- 4.3 Molecular Weight Determination of Proteins and Glycoproteins -- 4.3.1 Accuracy of Mass Determination -- 4.3.2 Sensitivity and Mass Range -- 4.4 Analysis of Oligonucleotides -- 4.5 Analysis of Glycans and Glycoconjugates -- 5 Combination of MALDI with Biochemical Methods -- 5.1 Peptide Mapping of Digested Proteins by MALDI -- 5.2 Combination of MALDI and Gel Electrophoresis -- 5.3 Combination of MALDI with Capillary Zone Electrophoresis -- 6 References -- III.5 MALDI Postsource Decay Mass Analysis -- 1 Introduction -- 2 Methodology and Principal Mechanisms of Postsource Decay -- 2.1 Internal Energy Uptake and Ion Stability -- 2.2 Instrumentation for MALDI-PSD Analysis -- 2.3 Precursor Ion Selection -- 3 Applications and Spectra Interpretation -- 3.1 Sequence Analysis of Peptides -- 3.2 Primary Structure Analysis of Unknown Peptides -- 5 References -- 3.3 Primary Structure Analysis of Modified Peptides and Other Biomolecules -- 4 Potential and Perspectives -- III.6 Electrospray Mass Spectrometry -- 1 Introduction -- 2 Instrumentation -- 2.1 The Electrospray Source -- 2.2 The Mass Analyzer -- 2.3 The Detector -- 3 Mass Spectra of Proteins -- 4 Coupling of Chromatographic Methods to the Mass Spectrometer -- 4.1 On-Line HPLC-MS. , 4.2 Coupling of a Protein Sequencer to an ESI Mass Spectrometer -- 4.3 Microcapillary LC Coupled to Mass Spectrometry -- 4.4 Capillary Electrophoresis Coupled to Mass Spectrometry -- 5 Purity Control of Synthetic Peptides -- 5.1 Sample Introduction with an Autosampler -- 5.2 Off-Line HPLC-MS -- 5.3 Characterization of Combinatorial Compound Collections -- 6 References -- III.7 Fourier-Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR-MS) -- 1 Introduction -- 2 Principle -- 2.1 Resolution, Mass Accuracy, Mass Range and Sensitivity -- 2.2 Tandem Mass Spectrometry -- 3 Electrospray and FT-ICR-MS -- 4 References -- III.8 Sequence Analysis of Proteins and Peptides by Mass Spectrometry -- 1 Introduction -- 2 Basics in Peptide Fragmentation -- 3 Instrumentation and Generation of MS/MS Data Sets -- 3.1 MALDI-PSD Time-of-Flight Mass Spectrometry -- 3.2 ESI-Triple Stage Quadrupole Mass Spectrometry -- 3.3 ESI-Ion Trap Mass Spectrometry -- 3.4 ESI-Quadrupole-TOF Mass Spectrometry -- 4 Coupling Methods for Mass Spectrometry -- 4.1 HPLC Mass Spectrometry -- 4.2 CZE Mass Spectrometry -- 4.3 Microchips -- 4.4 Nanospray -- 5 Identification of Posttranslational Modifications -- 5.1 Disulfide Bond Location -- 5.2 Other Posttranslational Modifications -- 6 Interpretation of Mass Spectrometric Data -- 6.1 Identification of Proteins by Database Search -- 6.2 De novo Sequencing of Peptides -- 7 References -- Section IV: Computer Sequence Analysis -- IV.1 Internet Resources for Protein Identification and Characterization -- 1 Introduction -- 2 General Approach -- 2.1 Experimental Attributes and Choice of Programs -- 2.2 Information in Databases -- 2.2.1 The Annotations in SWISS-PROT -- 2.2.2 SWISS-PROT Supplement TrEMBL -- 3 Identification and Characterization Tools at ExPASy -- 3.1 Identification Tools -- 3.1.1 Identification with Sequence Tags: TagIdent. , 3.1.2 Identification with Amino Acid Composition: AACompIdent.