How To Determine Amino Acid Sequence

Unraveling the amino acid sequence of a protein is akin to deciphering the blueprint of life itself. This precise order, also known as the primary structure, dictates a protein's unique three-dimensional shape and, consequently, its biological function. Determining this sequence is a cornerstone of biochemistry, molecular biology, and proteomics, enabling us to understand protein evolution, design novel therapeutics, and engineer enzymes with desired properties.

Why Determine Amino Acid Sequence?

Knowing the amino acid sequence of a protein offers a wealth of information:

• Understanding Protein Function: The sequence dictates how a protein folds and interacts with other molecules.
• Identifying Unknown Proteins: Comparing a newly determined sequence to databases can reveal its identity or similarity to known proteins.
• Studying Evolutionary Relationships: Sequence comparisons reveal how proteins have evolved over time, tracing evolutionary pathways.
• Designing Drugs and Therapies: Understanding a protein's structure allows for the design of drugs that specifically target and modulate its function.
• Engineering Proteins: Modifying the sequence can lead to proteins with enhanced stability, altered activity, or new functionalities.
• Diagnosing Diseases: Identifying mutations or sequence variations associated with diseases can aid in diagnosis and personalized medicine.

Classical Methods of Amino Acid Sequencing

Before the advent of modern proteomics techniques, determining amino acid sequences relied on laborious and time-consuming chemical methods. While largely superseded, understanding these classical methods provides valuable context and appreciation for the evolution of sequencing technologies.

1. Edman Degradation

The Edman degradation, developed by Pehr Edman, was a revolutionary method for sequentially removing and identifying amino acids from the N-terminus of a polypeptide chain. This technique became the workhorse of protein sequencing for several decades.

The Process:

1. N-terminal Modification: The protein is reacted with phenylisothiocyanate (PITC), which binds specifically to the N-terminal amino acid.
2. Cleavage: Under acidic conditions, the derivatized N-terminal amino acid is cleaved off as a phenylthiocarbamoyl (PTC) derivative. Crucially, the peptide bond between the first and second amino acid is selectively broken, leaving the rest of the polypeptide chain intact.
3. Identification: The PTC-amino acid is then identified using chromatography, such as High-Performance Liquid Chromatography (HPLC), by comparing its retention time to standards of known PTC-amino acids.
4. Repetition: The process is repeated on the shortened polypeptide chain, allowing for the sequential identification of amino acids.

Limitations:

• Length Limitations: The Edman degradation is most effective for sequencing relatively short peptides (typically up to 50-60 amino acids). Longer chains result in cumulative losses due to incomplete reactions and side reactions.
• N-terminal Blockage: The N-terminus of a protein must be free for the reaction to occur. Some proteins have blocked N-termini (e.g., acetylated or formylated), which must be removed before sequencing.
• Purity Requirements: The protein sample must be highly pure, as contaminants can interfere with the reaction and identification steps.

2. Chemical and Enzymatic Cleavage

Prior to Edman degradation, large proteins needed to be cleaved into smaller, manageable fragments. This was achieved through chemical and enzymatic methods:

• Chemical Cleavage:
  • Cyanogen Bromide (CNBr): Cleaves specifically at the carboxyl side of methionine residues. This is a widely used method as methionine is relatively rare in proteins.
  • Hydroxylamine: Cleaves specifically at Asn-Gly bonds.
• Enzymatic Cleavage (Proteases):
  • Trypsin: Cleaves at the carboxyl side of lysine and arginine residues, provided the adjacent residue is not proline.
  • Chymotrypsin: Cleaves at the carboxyl side of aromatic amino acids (phenylalanine, tyrosine, tryptophan), although with lower specificity than trypsin.
  • Pepsin: Cleaves preferentially at the amino side of leucine, phenylalanine, and tryptophan.
  • Thermolysin: Cleaves at the amino side of isoleucine, methionine, valine, leucine, and alanine.

Strategy:

The strategy involved cleaving the protein with different enzymes or chemical reagents, generating overlapping peptide fragments. These fragments were then separated, purified, and sequenced individually using Edman degradation. By comparing the overlapping sequences, the complete sequence of the original protein could be reconstructed. This was a complex and intricate process that required meticulous planning and execution.

3. Identification of N-terminal and C-terminal Amino Acids

Before complete sequencing became feasible, identifying the N-terminal and C-terminal amino acids provided valuable information about the protein.

• N-terminal Identification:
  • Sanger's Reagent (1-fluoro-2,4-dinitrobenzene, FDNB): Reacts with the free amino group of the N-terminal amino acid. The protein is then hydrolyzed, and the derivatized amino acid is identified by chromatography.
  • Dansyl Chloride (5-dimethylaminonaphthalene-1-sulfonyl chloride): Similar to Sanger's reagent but yields fluorescent derivatives, increasing sensitivity.
• C-terminal Identification:
  • Carboxypeptidases: Enzymes that sequentially cleave amino acids from the C-terminus of a polypeptide chain. Different carboxypeptidases have different specificities for the type of amino acid they release. By monitoring the release of amino acids over time, the C-terminal sequence can be deduced.
  • Hydrazine: Reacts with all peptide bonds, except the one involving the C-terminal carboxyl group, releasing all amino acids as aminoacyl hydrazides. The free C-terminal amino acid can then be identified.

Modern Methods of Amino Acid Sequencing: Mass Spectrometry

The advent of mass spectrometry (MS) revolutionized protein sequencing, offering speed, sensitivity, and the ability to analyze complex protein mixtures. MS-based proteomics has become the dominant approach for determining amino acid sequences.

1. Principles of Mass Spectrometry

Mass spectrometry measures the mass-to-charge ratio (m/z) of ions. A mass spectrometer consists of three main components:

1. Ion Source: Converts the sample molecules into gas-phase ions. Common ionization techniques include:
   • Electrospray Ionization (ESI): A gentle ionization method that is well-suited for large biomolecules like proteins. The sample is sprayed through a charged needle, forming droplets that evaporate, leaving multiply charged ions.
   • Matrix-Assisted Laser Desorption/Ionization (MALDI): The sample is mixed with a matrix compound and irradiated with a laser. The matrix absorbs the laser energy, causing the analyte molecules to be ionized and desorbed into the gas phase.
2. Mass Analyzer: Separates ions based on their m/z ratio. Different types of mass analyzers offer varying resolution, accuracy, and speed:
   • Quadrupole: Uses oscillating electric fields to filter ions based on their m/z ratio.
   • Time-of-Flight (TOF): Measures the time it takes for ions to travel through a flight tube. Ions with lower m/z ratios travel faster.
   • Ion Trap: Traps ions in a defined space using electric fields. Ions can be selectively ejected based on their m/z ratio.
   • Orbitrap: Measures the frequency of ion oscillation around a central electrode. This provides very high resolution and accuracy.
3. Detector: Detects the ions and measures their abundance. The detector generates a signal that is proportional to the number of ions hitting it.

2. Bottom-Up Proteomics (Shotgun Proteomics)

The most common MS-based approach for protein sequencing is bottom-up proteomics, also known as shotgun proteomics. In this method, proteins are digested into peptides before analysis by MS.

The Process:

1. Protein Digestion: The protein sample, which can be a complex mixture, is digested into peptides using a protease, typically trypsin. Trypsin's highly specific cleavage after lysine and arginine residues results in peptides with predictable lengths and C-terminal residues, facilitating sequence analysis.
2. Peptide Separation: The complex peptide mixture is separated using liquid chromatography (LC), often reversed-phase LC (RP-LC). This separates peptides based on their hydrophobicity, reducing the complexity of the sample entering the mass spectrometer.
3. Tandem Mass Spectrometry (MS/MS): The separated peptides are then analyzed by tandem mass spectrometry. In MS/MS, a selected peptide ion (precursor ion) is fragmented in a collision cell by colliding it with an inert gas (e.g., argon or nitrogen). This fragmentation generates a series of fragment ions, which are then analyzed by a second mass analyzer.
4. Fragment Ion Analysis: The pattern of fragment ions provides information about the amino acid sequence of the peptide. The most common fragmentation pathways result in b-ions (N-terminal fragments) and y-ions (C-terminal fragments). The mass difference between consecutive b-ions or y-ions corresponds to the mass of a specific amino acid.
5. Database Searching: The obtained MS/MS spectra are then compared to theoretical spectra generated from protein sequence databases. Algorithms identify the peptide sequence that best matches the observed fragmentation pattern. This process identifies the peptides present in the sample.
6. Protein Identification and Quantification: By identifying the peptides present in the sample, the proteins from which they originated can be inferred. Software tools are used to assemble the identified peptides into proteins and quantify their abundance.

Advantages of Bottom-Up Proteomics:

• High Throughput: Can analyze complex protein mixtures.
• High Sensitivity: Can detect low-abundance proteins.
• Automation: The process can be largely automated.

Limitations:

• Peptide Identification Challenges: Some peptides may be difficult to identify due to post-translational modifications, unusual amino acid compositions, or database limitations.
• Protein Coverage: Not all regions of a protein may be covered by identified peptides, potentially missing information about specific domains or modifications.
• Inference of Protein Sequence: The protein sequence is inferred from the identified peptides, which can be problematic for proteins with high sequence similarity.

3. De Novo Sequencing

In cases where a protein sequence is not present in the database, de novo sequencing can be used. This involves determining the amino acid sequence directly from the MS/MS spectrum without relying on a database.

The Process:

• Manual or Automated Interpretation: The MS/MS spectrum is carefully analyzed to identify the mass differences between fragment ions, which correspond to the masses of individual amino acids.
• Sequence Assembly: The amino acid sequence is assembled based on the identified mass differences and the known rules of peptide fragmentation.

Challenges:

• Complexity of Spectra: De novo sequencing can be challenging due to the complexity of MS/MS spectra, especially for modified peptides or peptides with unusual fragmentation patterns.
• Ambiguity: The assignment of amino acids can be ambiguous, especially for amino acids with similar masses (e.g., leucine and isoleucine).
• Requires Expertise: De novo sequencing requires significant expertise in mass spectrometry and peptide fragmentation.

4. Top-Down Proteomics

Top-down proteomics involves analyzing intact proteins directly by MS, without prior digestion. This approach preserves information about post-translational modifications and protein isoforms, which can be lost in bottom-up proteomics.

The Process:

1. Protein Separation: Intact proteins are separated using techniques such as two-dimensional gel electrophoresis (2D-PAGE) or LC.
2. Direct Infusion or LC-MS/MS: The separated proteins are then introduced directly into the mass spectrometer or analyzed by LC-MS/MS.
3. Protein Fragmentation: In the mass spectrometer, intact proteins are fragmented using techniques such as electron-transfer dissociation (ETD) or collision-induced dissociation (CID). ETD is particularly useful for top-down proteomics as it tends to preserve post-translational modifications.
4. Fragment Ion Analysis and Sequencing: The fragment ions are analyzed to determine the amino acid sequence and identify any post-translational modifications.

Advantages of Top-Down Proteomics:

• Preservation of Modifications: Retains information about post-translational modifications and protein isoforms.
• Complete Sequence Coverage: Provides complete sequence coverage of the protein.
• Identification of Protein Forms: Can identify different forms of a protein, such as splice variants or proteolytic fragments.

Limitations:

• Technical Challenges: Analyzing intact proteins by MS is technically challenging due to their large size and complexity.
• Lower Throughput: Top-down proteomics typically has lower throughput than bottom-up proteomics.
• Data Analysis Complexity: The analysis of top-down MS data is more complex than bottom-up data.

Strategies to Improve Amino Acid Sequencing

Several strategies can be employed to improve the accuracy and completeness of amino acid sequencing:

• Combining Different Proteases: Using multiple proteases with different cleavage specificities can generate overlapping peptide fragments, improving sequence coverage.
• Enrichment of Modified Peptides: Techniques such as immunoprecipitation or chemical derivatization can be used to enrich for peptides containing post-translational modifications, facilitating their identification.
• High-Resolution Mass Spectrometry: Using high-resolution mass spectrometers, such as Orbitrap instruments, improves the accuracy of mass measurements, reducing ambiguity in peptide identification and de novo sequencing.
• Advanced Fragmentation Techniques: Utilizing advanced fragmentation techniques, such as ETD and higher-energy collisional dissociation (HCD), can provide more comprehensive fragmentation patterns, improving sequence coverage and the identification of post-translational modifications.
• Improved Database Searching Algorithms: Developing more sophisticated database searching algorithms can improve the accuracy and sensitivity of peptide identification.
• Manual Validation: Manually validating peptide identifications and de novo sequence assignments can help to correct errors and improve the overall quality of the sequence data.

Applications of Amino Acid Sequencing

Amino acid sequencing has broad applications in various fields:

• Biotechnology: Protein engineering, antibody development, and production of recombinant proteins.
• Pharmaceuticals: Drug discovery, target identification, and personalized medicine.
• Diagnostics: Disease biomarker discovery and development of diagnostic assays.
• Food Science: Food safety, allergen detection, and quality control.
• Agriculture: Crop improvement and development of pest-resistant plants.
• Basic Research: Understanding protein structure-function relationships, studying evolutionary biology, and exploring the complexity of biological systems.

Conclusion

Determining the amino acid sequence of a protein is a fundamental task in modern biology. While classical methods like Edman degradation laid the groundwork, mass spectrometry-based proteomics has revolutionized the field, providing rapid, sensitive, and comprehensive sequence information. Bottom-up, top-down, and de novo sequencing approaches offer complementary strategies for analyzing proteins, each with its own strengths and limitations. As technology advances, amino acid sequencing will continue to play a critical role in understanding the complexities of life and developing new solutions for health, agriculture, and industry. The ability to accurately and efficiently determine protein sequences empowers researchers to unravel the intricate mechanisms of biological processes and opens doors to innovative applications across diverse fields.