Direct Protein Sequencing: Techniques and Applications

Direct Protein Sequencing

Overview of Protein Sequencing

Direct protein sequencing is a fundamental technique in biochemistry used to determine the order of amino acids in a protein. The primary structure of a protein dictates all other levels of structure and function, making its determination essential for understanding protein biology.

• Primary structure: The linear sequence of amino acids in a protein.

• Protein sequencing: The process of determining the order of amino acids from the N-terminus to the C-terminus of a protein.

• Importance: Knowing the sequence allows for prediction of the 3D structure and function of the protein.

Problems in protein analysis: Large proteins must be cleaved or fragmented into smaller peptides for sequencing.

General Steps in Protein Sequencing

1. Cell Lysis: Breaking open cells to release proteins.

2. Protein Purification: Isolating the protein of interest from the mixture.

3. Fragmentation: Cleaving the protein into smaller peptides using chemical or enzymatic methods.

4. Sequencing: Determining the amino acid sequence of the peptides.

5. Assembly: Piecing together the peptide sequences to reconstruct the full protein sequence.

Example Workflow

• Proteins are extracted from cells, purified, and then fragmented.

• Fragments are sequenced, and the data is used to deduce the original protein sequence.

Techniques to Sequence a Protein

Protein Cleavage Techniques

Several methods are used to cleave proteins into smaller fragments for sequencing. Each method targets specific peptide bonds and yields characteristic products.

Technique	Reagents	Peptide Bonds Cleaved	Result
Amino Acid Hydrolysis	6M HCl	All peptide bonds	Free amino acids
Chemical Cleavage	Specific chemicals (e.g., cyanogen bromide)	Chemical-specific	Peptide fragments
Peptidases	Enzymes (e.g., trypsin, chymotrypsin)	Enzyme-specific	Peptide fragments
Edman Degradation	Phenylisothiocyanate (PITC)	N-terminal amino acid	Sequential protein sequencing

Key Protein Sequencing Methods

• Amino Acid Hydrolysis: Uses strong acid (6M HCl) to break all peptide bonds, releasing free amino acids. Used to determine amino acid composition, not sequence.

• Chemical Cleavage: Uses chemicals like cyanogen bromide to cleave at specific residues (e.g., methionine). Produces predictable peptide fragments.

• Enzymatic Cleavage: Uses proteases (e.g., trypsin, chymotrypsin) to cleave at specific amino acid residues, generating fragments for sequencing.

• Edman Degradation: Sequentially removes one amino acid at a time from the N-terminus, allowing direct determination of the sequence.

Examples and Applications

• Edman Degradation is widely used for sequencing short peptides and proteins.

• Chemical and enzymatic cleavage are often combined to generate overlapping fragments for complete sequence determination.

Practice Questions (from notes)

• Which of the following is a protein sequencing technique?

  • a) Amino acid hydrolysis

  • b) Edman Degradation

  • c) Peptidase/enzymatic cleavage

Matching Techniques to Descriptions

• Chemical cleavage (e.g., cyanogen bromide): Cleaves peptide bonds at specific residues (e.g., methionine).

• Edman Degradation: Sequentially removes N-terminal amino acids for direct sequencing.

• Enzymatic cleavage (e.g., trypsin): Uses proteases to cleave at specific amino acid residues.

• 6M HCl: Used in amino acid hydrolysis to break all peptide bonds, yielding free amino acids.

Summary Table: Protein Sequencing Techniques

Method	Hydrolyzes	Reagent/Enzyme	Result
Amino Acid Hydrolysis	All peptide bonds	6M HCl	Free amino acids
Chemical Cleavage	Specific peptide bonds	Chemical (e.g., cyanogen bromide)	Peptide fragments
Enzymatic Cleavage	Specific peptide bonds	Protease (e.g., trypsin)	Peptide fragments
Edman Degradation	N-terminal amino acid	PITC	Sequential amino acid identification

Key Equations and Concepts

• Hydrolysis Reaction: $\text{Peptide bond} + H_2O \xrightarrow{\text{6M HCl}} \text{Amino acids}$

• Edman Degradation Reaction: $\text{PITC} + \text{N-terminal amino acid} \rightarrow \text{PTH-amino acid (identified)} + \text{shortened peptide}$

Additional info: Edman Degradation is most effective for peptides up to about 50 residues. For larger proteins, fragmentation is necessary before sequencing.