Protein Primary Structure, Purification and Sequencing
Page 1: Amino Acid Sequences of Cytochrome
Table displaying amino acid sequences of Cytochrome c from 38 species
Importance for understanding evolutionary relationships based on sequence variations
Page 2: Learning Goals
Learn the chemistry of peptide bond formation
Understand structural properties of the peptide bond
Basics of protein purification
Techniques of protein sequencing
Discuss uses of protein sequences
Page 3: Formation of Peptides
Peptides: small condensation products of amino acids
Characterization: small compared to proteins (Molecular weight < 10 kDa)
Page 4: Structure of Peptides and Proteins
Linear sequence of amino acids
Each peptide has a free N-terminal and C-terminal
Amino acids are linked by peptide bonds
Sequence convention: written from N to C terminus
Page 5: Why We Bother
Catalysis: Examples include enolase and DNA polymerase
Transport: Hemoglobin for O2 transport and lactose permease for transporting lactose
Structure: Collagen and keratin provide structural integrity in various tissues
Motion: Myosin and actin facilitate muscle contraction and cell motility
Page 6: Proteomics
Understanding disease mechanisms through protein functions and interactions
Relationship between genome, transcriptome, and proteome
30,000 genes encoding for > 500,000 proteins, highlighting complexity
Page 7: What to Study About Peptides and Proteins?
Sequence and composition: What are the amino acids present?
Three-dimensional structure: Conformation related to function
Native fold: Mechanisms by which proteins achieve their stable forms
Functionality: How proteins perform their biological roles
Interactions: Peptide interactions with other macromolecules
Cell localization and physico-chemical properties
Page 8: Covalent Structures of Proteins
Sequence determination techniques:
Gene sequence: Translations from DNA to protein
Chemical/enzymatic sequencing of purified proteins including mass spectrometry
Page 9: Protein Purification Basics
Knowledge of gene sequence is essential
Understanding protein properties: size, charge, structure, function
Design purification schemes accordingly
Page 10: Protein Purification Techniques
When protein function is known, but sequence is not:
Utilize various separation techniques
Monitor concentrations and functions during purification
Page 11: Separation Principles
Based on physico-chemical properties:
Size
Charge
Ligand affinity
Solubility
Hydrophobicity
Thermal stability
Chromatography: Commonly employed preparative separation
Page 12: Assaying for Function
Need an assay to determine protein function and concentration
Utilize UV absorbance techniques and Beer-Lambert Law:
C = A/εl where A is absorbance, ε is extinction coefficient, l is path length
Page 13: Protein Molar Mass Examples
Insulin: 5.8 kD
Cytochrome C: ~12 kD
Ribonuclease: ~13.7 kD
Lysozyme: ~14.7 kD
Myoglobin: ~17 kD
Hemoglobin: ~68 kD
Immunoglobulin: ~150-200 kD
Glutamine synthetase: ~400 kD
Page 14: Ammonium Sulfate Precipitation
Increases ionic strength, proteins precipitate as hydration shells are disrupted
Larger proteins precipitate at lower ammonium sulfate concentrations
Page 15: Ammonium Sulfate Precipitation Overview
Typically the first purification step
Removes large batches of contaminants
May require further purification
Page 16: Effects of pH on Solubility
At pH = pI, proteins precipitate due to loss of charge repulsion
Thermal stability influences precipitation during unfolding
Page 17: Size-Based Separation Techniques
Gel filtration and size exclusion chromatography:
Varies based on sizes of molecules,
Small molecules are retained in the gel, larger molecules pass through
Page 18: Size Exclusion Limits
Specific size ranges for each resin:
3000 – 15,000
50,000 – 500,000
10,000 – 100,000
Proteins below/exceeding limits elute at bed/void volume
Page 19: Elution Profile Analysis
Understanding elution profiles from size exclusion columns for various proteins
Page 20: Further Elution Profiles
Evaluate separation of proteins across ranges, detail properties of each
Page 21: Additional Elution Profiles
Continue analyzing separation based on given exclusion sizes and protein attributes
Page 22: Understanding Charge
Isoelectric point (pI): pH where protein net charge is zero
Charge transitions: below pI (positive), above pI (negative)
Page 23: Calculating pI of Peptides
Use of chemical structures and pKa values to determine pI
Page 24: Determining pI with Experiments
Utilize tools like expasy site to calculate pI
Relationship of amino acid composition to pI values
Page 25: Ion Exchange Resins
Charged ion exchange resins utilized for separation based on pI and pH
Page 26: Binding Properties of Resins
Negatively charged proteins bind to positively charged anion exchange resins
Proprietary designs: DEAE and Q sepharose
Page 27: Cation Exchange Resins
Positively charged proteins bind to negatively charged cation exchange resins
Examples: CM and SP resins
Page 28: Protein Elution Strategies
Methods to elute bound proteins: changing pH or salt concentration
Page 29: Elution Profile for Cation Exchange Column
Analyze interactions and elution conditions for proteins of varying sizes and charges
Page 30: Hypothetical Protein Binding Analysis
Assumptions about protein sequences based on elution properties
Page 31: Affinity Chromatography
Utilizing specific interactions, such as ATP columns to purify binding proteins
Page 32: Binding Mechanism
Process by which proteins bind to the column and methods to elute bound proteins
Page 33: Polyacrylamide Gel Electrophoresis (
Size-dependent separation technique
SDS treatment used for denaturation and dissociation of multi-subunit proteins
Page 34: SDS PAGE
Sodium dodecyl sulfate binds proteins and imparts a negative charge
Size is the primary determinant for migration speed
Page 35: Importance of Protein Characterization
Characterizing proteins aids understanding of function, structure, and potential therapeutic applications
Page 36: Examples of Protein Structures
Illustrate complexity of protein structures from primary to quaternary
Page 37: Primary Structure of Insulin
Variability in protein sizes and methodologies for sequence determination
Page 38: Quantitative Amino Acid Analysis
Steps undertaken in analysis include hydrolyzing amide bonds and utilizing ion exchange chromatography
Page 39: Chemical Derivatization for Analysis
DABS used for enhancing detection of amino acids after hydrolysis
Page 40: Caution in Amino Acid Analysis
Importance of understanding conversion of specific residues during hydrolysis
Page 41: End-Group Analysis
Techniques to determine identities of N-terminal and C-terminal amino acids
Page 42: N-Terminal Reagents Overview
Agents that react with free amino groups; for example, dansyl chloride or FDNB
Page 43: Methodology for N-Terminal Analysis
Techniques involving dansyl chloride to identify amino acids at the N-terminus
Page 44: Use of Standards in Analysis
Standards assist in identifying labeled amino acids and determining protein heterogeneity.
Page 45: Disulfide Bonds and Sequencing
Presence of disulfide bonds affects sequencing methods and outcomes.
Page 46: Breaking Disulfide Bonds
Reagents like β-mercaptoethanol and DTT used for disulfide bond cleavage.
Page 47: Reaction of Reducing Agents
Specific molecular processes involved in reduction of cystine to cysteine.
Page 48: DTT in Disulfide Bond Cleavage
DTT forms cyclic disulfides through disulfide exchange processes.
Page 49: Carboxymethylation Technique
Prevents reformation of disulfide bonds during sequencing processes.
Page 50: Protein Sequencing Challenges
Techniques best suited for fragments of 30-50 amino acids; utilize proteases for effective fragmentation.
Page 51: Protein Sequencing Procedures
Steps include cleavage, sequencing fragments, and analyzing data for complete protein sequence.
Page 52: Summary of Hydrolyzing and Sequencing Steps
Overview of the methodologies and expected outcomes during sequencing experiments.
Page 53: Cleavage Methods for Proteins
Enzymatic/chemical methods employed to generate peptide fragments for further analysis.
Page 54: Peptide Fragmentation Examples
Illustrate typical peptide sequences produced by trypsin and chymotrypsin digestion.
Page 55: Chemical Peptide Generation
Cyanogen bromide's specificity in cleavage at methionine residues.
Page 56: Edman Degradation Methodology
Discuss efficiency and limitations of Edman degradation in peptide sequencing.
Page 57: Edman Method Limitations
Factors affecting efficacy such as signal-to-noise ratio with longer sequences.
Page 58: Comprehensive Protein Sequencing Steps
Procedure for comprehensive sequencing of a protein involving multiple methodologies.
Page 59: Data Analysis Techniques
Effective strategies for comparing and analyzing peptide fragments post-sequencing.
Page 60: Eliminating Redundancies in Data
Identify and remove duplicate fragments for clearer data interpretation.
Page 61: Fragment Overlapping Techniques
Strategies for aligning overlapping fragments to reconstruct sequences.
Page 62: Modern Sequencing Techniques
The process of isolation and DNA sequencing linked to peptide/protein sequencing.
Page 63: Disulfide Bond Identification Techniques
Methods to distinguish fragments pre/post disulfide cleavage.
Page 64: Mass Spectrometry Applications
Use of electrospray ionization mass spectrometry to determine protein mass.
Page 65: Mass Spectrum Interpretation
Understanding the peaks in mass spectra concerning protein mass-to-charge ratios.
Page 66: Solving Mass Analysis Equations
Mathematical approach for analyzing mass/charge data for sequences.
Page 67: Mass Spectrum Data Presentation
Showcasing mass spectrum values for protein identification purposes.
Page 68: Mass Spectrometry in Sequencing
Peptide fragmentation followed by mass spectrometric analysis enhances sequence data.
Page 69: Overview of Tandem Mass Spectrometry
Processes involved in mass spectrometric analysis to further define protein sequences.
Page 70: Interpretation of m/z Values
Analyzing m/z values to identify specific amino acid sequences and their properties.
Page 71: Importance of Protein Sequences
Sequences assist in protein identification and understanding biological roles and functions.
Page 72: Evolutionary Relationships Through Sequencing
Insights into evolutionary relationships by comparing protein primary structures.
Page 73: Peptide Mapping for Mutations
Methodology for identifying mutations through peptide mapping techniques.
Page 74: Examples of Mutant Proteins
Use of hemoglobin examples to illustrate peptide mapping and mutation identification.
Page 75: 2D Electrophoresis Techniques
Dual experimental approaches for identifying protein differences.
Page 76: Sequence Searching Resources
Overview of available tools and databases for sequence alignment and comparison.