Principles of proteomics

Chapter 1 The origin and scope of proteomics. 1.2 THE BIRTH OF LARGE-SCALE BIOLOGY AND THE "OMICS" ERA ; 1.3 THE GENOME, TRANSCRIPTOME, PROTEOME, AND METABOLOME ; 1.4 FUNCTIONAL GENOMICS ; Transcriptomics is the systematic, global analysis of mRNA ; Large-scale mutagenesis and interference can also determine the functions of genes on a global scale ; 1.5 THE NEED FOR PROTEOMICS ; 1.6 THE SCOPE OF PROTEOMICS ; Protein identification and quantitation are the most fundamental aspects of proteomic analysis ; Important functional data can be gained from sequence and structural analysis ; Interaction proteomics and activity-based proteomics can help to link proteins into functional networks ; 1.7 CURRENT CHALLENGES IN PROTEOMICS Chapter 2 Strategies for protein separation. 2.2 GENERAL PRINCIPLES OF PROTEIN SEPARATION IN PROTEOMICS ; 2.3 PRINCIPLES FOR TWO-DIMENSIONAL GEL ELECTROPHORESIS ; Electrophoresis separates proteins by mass and charge ; Isoelectric focusing separates proteins by charge irrespective of mass ; SDS-PAGE separates proteins by mass irrespective of charge ; 2.4 THE APPLICATION OF 2DGE IN PROTEOMICS ; The four major advantages of 2DGE are robustness, reproducibility, visualization, and compatibility with downstream microanalysis ; The four major limitations of 2DGE are resolution, sensitivity, representation, and compatibility with automated protein analysis ; The resolution of 2DGE can be improved with giant gels, zoom gels, and modified gradients, or by pre-fractionating the sample ; The sensitivity of 2DGE depends on the visualization of minor protein spots, which can be masked by abundant proteins ; The representation of hydrophobic proteins is an intractable problem reflecting the buffers required for isoelectric focusing ; Downstream mass spectrometry requires spot analysis and picking ; 2.5 PRINCIPLES OF MULTIDIMENSIONAL LIQUID CHROMATOGRAPHY ; Protein and peptide separation by chromatography relies on differing affinity for stationary and mobile phases ; Affinity chromatography exploits the specific binding characteristics of proteins and/or peptides ; Size exclusion chromatography sieves molecules on the basis of their size ; Ion exchange chromatography exploits differences in net charge ; Reversed-phase chromatography and hydrophobic interaction chromatography exploit the affinity between peptides and hydrophobic resins ; 2.6 MULTIDIMENSIONAL LIQUID CHROMATOGRAPHY STRATEGIES IN PROTEOMICS ; Multidimensional liquid chromatography is more versatile and more easily automated than 2DGE but lacks a visual dimension ; The most useful MDLC systems achieved optimal peak capacity by exploiting orthogonal separations that have internally compatible buffers ; MudPIT shows how MDLC has evolved from a laborious technique to virtually hands-free operation ; RP-RPLC and HILIC-RP systems offer advantages for the separation of certain types of peptide mixtures ; Affinity chromatography is combined with MDLC to achieve the simplification of peptide mixtures Chapter 3 Strategies for protein identification. 3.2 PROTEIN IDENTIFICATION WITH ANTIBODIES ; 3.3 DETERMINATION OF PROTEIN SEQUENCES BY CHEMICAL DEGRADATION ; Complete hydrolysis allows protein sequences to be inferred from the content of the resulting amino acid pool ; Edman degradation was the first general method for the de novo sequencing of proteins ; Edman degradation was the first protein identification method to be applied in proteomics, but it is difficult to apply on a large scale ; 3.4 MASS SPECTROMETRY BASIC PRINCIPLES AND INSTRUMENTATION ; Mass spectrometry is based on the separation of molecules according to their mass/charge ratio ; The integration of mass spectrometry into proteomics required the development of soft ionization methods of prevent random fragmentation ; Controlled fragmentation is used to break peptide bonds and generate fragment ions ; Five principal types of mass analyzer are commonly used in proteomics ; 3.5 PROTEIN IDENTIFICATION USING DATA FROM MASS SPECTRA ; Peptide mass fingerprinting correlates experimental and theoretical intact peptide masses ; Shotgun proteomics can be combined with database searches based on uninterpreted spectra ; MS/MS spectra can be used to derive protein sequences de novo Chapter 4 Strategies for protein quantitation. 4.2 QUANTITATIVE PROTEOMICS BASED ON 2DGE ; The quantitation of proteins in two-dimensional gels involves the creation of digital data from analog images ; Spot detection, quantitation, and comparison can be challenging without human intervention ; 4.3 MULTIPLEXED IN-GEL PROTEOMICS ; Difference in-gel electrophoresis involves the simultaneous separation of comparative protein samples labeled with different fluorophores ; Parallel analysis with multiple dyes can also be used to identify particular structural or functional groups of proteins ; 4.4 QUANTITATIVE MASS SPECTROMETRY ; Label-free quantitation may be based on spectral counting or the comparison of signal intensities across samples in a narrow m/z range ; Label-based quantitation involves the incorporation of labels that allow corresponding peptides in different samples t be identified by a specific change in mass ; ICAT reagents are used for the selective labeling of proteins or peptides ; Proteins and peptides can also be labeled nonselectively ; Isobaric tagging allows protein quantitation by the detection of reporter ions ; Metabolic labeling introduces the label before sample preparation but is limited to simple organisms and cultured cells

Chapter 5 The analysis of protein sequences. 5.2 PROTEIN FAMILIES AND EVOLUTIONARY RELATIONSHIPS ; Evolutionary relationships between proteins are based on homology ; The function of a protein can often be predicted from its sequence ; 5.3 PRINCIPLES OF PROTEIN SEQUENCE COMPARISON ; Protein sequences can be compared in terms of identity and similarity ; Homologous sequences are found by pairwise similarity searching ; Substitution score matrices rank the importance of different substitutions ; Sequence alignment scores depend on sequence lengths ; Multiple alignments provide more information about key sequence elements ; 5.4 STRATEGIES TO FIND MORE DISTANT RELATIONSHIPS ; PSI-BLAST uses sequence profiles to carry out iterative searches ; Pattern recognition methods incorporate conserved sequence signatures ; 5.5 THE RISK OF FALSE-POSITIVE ANNOTATIONS Chapter 6 The analysis of protein structures. 6.2 STRUCTURAL GENOMICS AND STRUCTURE SPACE ; Coverage of structure space is currently uneven; Structure and function are not always related ; 6.3 TECHNIQUES FOR SOLVING PROTEIN STRUCTURES ; X-ray diffraction requires well-ordered protein crystals ; NMR spectroscopy exploits the magnetic properties of certain atomic nuclei ; Additional methods for structural analysis mainly provide supporting data ; 6.4 PROTEIN STRUCTURE PREDICTION ; Structural predictions can bridge the gap between sequence and structure ; Protein secondary structures can be predicted from sequence data ; Tertiary structures can be predicted by comparative modeling if a template structure is available ; Ab initio prediction methods attempt to construct structures from first principles ; Fold recognition (threading) is based on similarities between nonhomologous folds ; 6.5 COMPARISON OF PROTEIN STRUCTURES ; 6.6 STRUCTURAL CLASSIFICATION OF PROTEINS ; 6.7 GLOBAL STRUCTURAL GENOMICS INITIATIVES Chapter 7 Interaction proteomics. 7.2 METHODS TO STUDY PROTEIN-PROTEIN INTERACTIONS ; Genetic methods suggest interactions from the combined effects of two mutations in the same cell or organism ; Protein interactions can be suggested by comparative genomics and homology transfer ; Affinity-based biochemical methods provide direct evidence that proteins can interact ; Interactions between proteins in vitro and in vivo can be established by resonance energy transfer ; Surface plasmon resonance can indicate the mass of interaction proteins ; 7.3 LIBRARY-BASED METHODS FOR THE GLOBAL ANALYSIS OF BINARY INTERACTIONS ; 7.4 TWO-HYBRID/PROTEIN COMPLEMENTATION ASSAYS ; The yeast two-hybrid systems works by assembling a transcription factor from two inactive fusion proteins ; Several large-scale interaction screens have been carried out using different yeast two-hybrid screening strategies ; Conventional yeast two-hybrid screens have a significant error rate ; 7.5 MODIFIED TWO-HYBRID SYSTEMS FOR MEMBRANE, CYTOSOLIC, AND EXTRACELLULAR PROTEINS ; 7.6 BACTERIAL AND MAMMALIAN TWO-HYBRID SYSTEMS ; 7.7 LUMIER AND MAPPIT HIGH-THROUGHPUT TWO-HYBRID PLATFORMS ; 7.8 ADAPTED HYBRID ASSAYS FOR DIFFERENT TYPES OF INTERACTIONS ; 7.9 SYSTEMATIC COMPLEX ANALYSIS BY TANDEM AFFINITY PURIFICATION-MASS SPECTROMETRY ; 7.10 ANALYSIS OF PROTEIN INTERACTION DATA ; 7.11 PROTEIN INTERACTION MAPS ; 7.12 PROTEIN INTERACTIONS WITH SMALL MOLECULES

Chapter 8 Protein modification in proteomics. 8.2 METHODS FOR THE DETECTION OF POST-TRANSLATIONAL MODIFICATION ; 8.3 ENRICHMENT STRATEGIES FOR MODIFIED PROTEINS AND PEPTIDES ; 8.4 PHOSPHOPROTEOMICS ; Protein phosphorylation is a key regulatory mechanism ; Separated phosphorproteins can be detected with specific staining reagents ; Sample preparation for phosphoprotein analysis typically involves enrichment using antibodies or strongly cationic chromatography resins ; 8.5 ANALYSIS OF PHOSPHOPROTEINS BY MASS SPECTROMETRY ; A combination of Edman degradation and mass spectrometry can be used to map phosphorylation sites ; Intact phosphopeptide ions can be identified by MALDI-TOF mass spectrometry ; Phosphopeptides yield diagnostic marker ions and neutral loss products ; 8.6 QUANTITATIVE ANALYSIS OF ; 8.7 GLYCOPROTEOMICS ; Glycoproteins represent more than half of the eukaryotic proteome ; Glycans play important roles in protein stability, activity, and localization, and are important indicators of disease ; Conventional glycoanalysis involves the use of enzymes that remove specific glycan groups and the separation of glycoproteins by electrophoresis ; Glycoprotein-specific staining allows the glycoprotein to be studied by 2DGE ; There are two principal methods for glycoprotein enrichment that have complementary uses ; Mass spectrometry is used for the high-throughput identification and characterization of glycoprotein

Chapter 9 Protein microarrays. 9.2 THE EVOLUTION OF PROTEIN MICROARRAYS ; 9.3 DIFFERENT TYPES OF PROTEIN MICROARRAYS ; Analytical, functional, and reverse microarrays are distinguished by their purpose and the nature of the interacting components ; Analytical microarrays contain antibodies or other capture reagents ; Functional protein microarrays can be used to study a wide range of biochemical functions ; 9.4 THE MANUFACTURE OF FUNCTIONAL PROTEIN MICROARRAYS

PROTEIN SYNTHESIS ; Proteins can be synthesized by the parallel construction of many expression vectors ; Cell-free expression systems allow the direct synthesis of protein arrays in situ ; 9.5 THE MANUFACTURE OF FUNCTIONAL PROTEIN MICROARRAYS

PROTEIN IMMOBILIZATION ; 9.6 THE DETECTION OF PROTEINS ON MICROARRAYS ; Methods that require labels can involve either direct or indirect detection ; Label-free methods do not affect the intrinsic properties of interacting proteins ; 9.7 EMERGING PROTEIN CHIP TECHNOLOGIES ; Bead and particle arrays in solution represent the next generation of protein microarrays ; Cell and tissue arrays allow the direct analysis of proteins in vivo

Chapter 10 Applications of proteomics. 10.2 DIAGNOSTIC APPLICATIONS OF PROTEOMICS ; Proteomics is used to identify biomarkers of disease states ; Biomarkers can be discovered by finding plus/minus or quantitative differences between samples ; More sensitive techniques can be used to identify biomarker profiles ; 10.3 APPLICATIONS OF PROTEOMICS IN DRUG DEVELOPMENT ; Proteomics can help to select drug targets and develop lead compounds ; Proteomics is also useful for target validation ; Chemical proteomics can be used to select and develop lead compounds ; Proteomics can be used to assess drug toxicity during clinical development ; 10.4 PROTEOMICS IN AGRICULTURE ; Proteomics provides novel markers in plant breeding and genetics ; Proteomics can be used for the analysis of genetically modified plants ; 10.5 PROTEOMICS IN INDUSTRY

IMPROVING THE YIELD OF SECONDARY METABOLISM.