Understanding Peptide Sequences and Nomenclature

Understanding Peptide Sequences and Nomenclature

Peptide sequences are the fundamental language of peptide chemistry and biology. Accurate understanding and interpretation of these sequences are crucial for successful research and the development of reliable applications. This article provides a comprehensive overview of peptide sequences, nomenclature, and key considerations for quality assessment and sourcing.

Peptide Structure and Formation

Peptides are short chains of amino acids linked together by peptide bonds. These bonds are formed through a condensation reaction between the carboxyl group (-COOH) of one amino acid and the amino group (-NH₂) of another, releasing a water molecule (H₂O). The sequence of amino acids dictates the peptide's properties and function.

Each amino acid residue within a peptide has an N-terminal (amino terminus) and a C-terminal (carboxyl terminus). By convention, peptide sequences are written from the N-terminus to the C-terminus. For example, the sequence Ala-Gly-Ser represents a tripeptide where alanine (Ala) is the N-terminal residue and serine (Ser) is the C-terminal residue.

Amino Acid Nomenclature and Abbreviations

Each of the 20 common amino acids found in proteins has a unique name, three-letter abbreviation, and single-letter code. Using the correct nomenclature is essential for clear communication and accurate representation of peptide sequences.

Here's a table summarizing the standard amino acids:

When the sequence is known but a specific residue is uncertain, "X" is often used as a placeholder. For example, a protease cleavage site might be represented as P-X-P, indicating proline followed by an unknown amino acid, followed by proline.

Modified Amino Acids and Non-Standard Residues

In addition to the 20 standard amino acids, peptides can contain modified amino acids or non-standard residues. These modifications can significantly alter the peptide's properties and function. Common modifications include:

• Phosphorylation: Addition of a phosphate group (e.g., pSer, pThr, pTyr). Phosphorylation is a crucial regulatory mechanism in many biological processes.
• Acetylation: Addition of an acetyl group (e.g., Ac-Lys). Acetylation often occurs at the N-terminus or on lysine residues and can affect protein-protein interactions and stability.
• Amidation: Conversion of the C-terminal carboxyl group to an amide (e.g., -NH₂). C-terminal amidation neutralizes the negative charge of the C-terminus, potentially affecting interactions.
• Glycosylation: Addition of a carbohydrate moiety (e.g., N-glycosylation on Asn, O-glycosylation on Ser/Thr). Glycosylation affects folding, stability, and interactions.
• Disulfide Bridges: Formation of a covalent bond between two cysteine residues. Disulfide bridges stabilize the peptide's three-dimensional structure.
• D-Amino Acids: Isomers of the naturally occurring L-amino acids. Incorporation of D-amino acids can increase resistance to enzymatic degradation.

When ordering peptides with modified amino acids, it's critical to specify the modification clearly and unambiguously. Consult with the peptide synthesis vendor to ensure accurate incorporation of the desired modification.

Peptide Synthesis and Purity

Peptides are typically synthesized using solid-phase peptide synthesis (SPPS). This method involves stepwise addition of amino acids to a growing peptide chain attached to a solid support (resin). After synthesis, the peptide is cleaved from the resin and purified.

Purity is a critical parameter in peptide quality. It refers to the percentage of the peptide that is the desired sequence. Typical purity levels range from crude (<70%) to high purity (>98%).

Factors affecting peptide purity:

• Incomplete coupling: Not all amino acids react completely during each coupling step, leading to deletion sequences.
• Side-chain protecting group removal: Incomplete removal of protecting groups can result in modified peptides.
• Racemization: Conversion of L-amino acids to D-amino acids, especially during coupling of sterically hindered amino acids.
• Aggregation: Peptides can aggregate during synthesis, hindering efficient reactions.

Practical Tip: For most research applications, a purity level of 80-95% is sufficient. However, for quantitative assays or applications requiring high specificity, higher purity (>95%) is recommended.

Peptide Characterization and Quality Control

Several analytical techniques are used to characterize peptides and assess their quality:

• HPLC (High-Performance Liquid Chromatography): Separates peptides based on their hydrophobicity. HPLC is used to determine peptide purity and identify impurities. A typical HPLC chromatogram should show a single, sharp peak for a highly pure peptide.
• Mass Spectrometry (MS): Determines the mass-to-charge ratio (m/z) of the peptide. MS is used to confirm the correct molecular weight and identify any modifications or unexpected byproducts. Common MS techniques include MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight) and ESI (Electrospray Ionization).
• Amino Acid Analysis (AAA): Determines the amino acid composition of the peptide. AAA is used to verify the correct amino acid ratios and identify any errors in the sequence.
• Peptide Sequencing (Edman Degradation): Sequentially removes and identifies amino acids from the N-terminus. Edman degradation is used to confirm the peptide sequence, but it is less commonly used now due to the prevalence of MS.

Practical Tip: Request HPLC and MS data from your peptide vendor. Compare the reported molecular weight with the theoretical molecular weight calculated from the sequence. A mass difference of more than +/- 1 Da (Dalton) may indicate a modification or error in synthesis.

Peptide Solubility and Handling

Peptide solubility is influenced by the amino acid composition and sequence. Hydrophobic peptides tend to be less soluble in aqueous solutions, while peptides with charged residues (Asp, Glu, Lys, Arg) are generally more soluble.

Factors affecting peptide solubility:

• Hydrophobicity: Peptides with a high proportion of hydrophobic amino acids (Ala, Val, Leu, Ile, Phe, Trp) may require organic solvents for dissolution.
• pH: Adjusting the pH can improve solubility by protonating or deprotonating charged residues. Acidic peptides (containing Asp or Glu) are more soluble at higher pH, while basic peptides (containing Lys or Arg) are more soluble at lower pH.
• Salt concentration: Adding a small amount of salt (e.g., NaCl) can help to disrupt peptide aggregates and improve solubility.

Solubility Guidelines:

• For peptides with a net positive charge, try dissolving in water with a small amount of acetic acid (0.1-1%).
• For peptides with a net negative charge, try dissolving in water with a small amount of ammonium hydroxide (0.1-1%).
• For highly hydrophobic peptides, try dissolving in organic solvents such as DMSO or DMF. Add the organic solvent dropwise to the peptide, vortexing continuously, until the peptide is completely dissolved. Then, dilute the solution with water or buffer to the desired concentration. The final concentration of organic solvent should be kept as low as possible to avoid adverse effects on downstream applications.

Practical Tip: Start by dissolving the peptide in a small volume of solvent. If the peptide does not dissolve readily, try sonicating or heating the solution gently. Avoid prolonged heating, as this can lead to degradation.

Peptide Stability and Storage

Peptides are susceptible to degradation through various mechanisms, including oxidation, hydrolysis, and microbial contamination. Proper storage is essential to maintain peptide integrity.

Factors affecting peptide stability:

• Temperature: Higher temperatures accelerate degradation.
• Moisture: Moisture promotes hydrolysis.
• Light: Exposure to light can cause oxidation.
• pH: Extreme pH values can promote hydrolysis.
• Proteases: Proteases can degrade peptides.

Storage Recommendations:

• Store peptides in a tightly sealed container under anhydrous conditions (e.g., with a desiccant).
• Store peptides at -20°C or -80°C.
• Avoid repeated freeze-thaw cycles. Aliquot the peptide into smaller portions to minimize freeze-thaw cycles.
• Dissolve peptides in a buffer solution with a pH near neutral (pH 6-8).
• Add protease inhibitors to the buffer solution to prevent degradation by proteases.
• Protect peptides from light by storing them in amber vials or wrapping them in aluminum foil.

Sourcing Considerations

Choosing a reliable peptide vendor is crucial for obtaining high-quality peptides. Consider the following factors when selecting a vendor:

• Experience and reputation: Choose a vendor with a proven track record of producing high-quality peptides.
• Synthesis capabilities: Ensure that the vendor can synthesize peptides with the desired length, purity, and modifications.
• Quality control: Verify that the vendor performs comprehensive quality control testing, including HPLC, MS, and AAA.
• Pricing: Compare prices from multiple vendors. Be wary of unusually low prices, as this may indicate compromised quality.
• Customer service: Choose a vendor with responsive and helpful customer service.

Practical Tip: Request a certificate of analysis (CoA) from the vendor for each peptide. The CoA should include the peptide sequence, purity, molecular weight, and HPLC and MS data. Review the CoA carefully to ensure that the peptide meets your requirements.

Key Takeaways

• Peptide sequences are written from the N-terminus to the C-terminus.
• Use the correct amino acid nomenclature (three-letter or single-letter codes).
• Modified amino acids can significantly alter peptide properties.
• Peptide purity is a critical parameter. Request HPLC and MS data from your vendor.
• Proper storage is essential to maintain peptide integrity. Store peptides at -20°C or -80°C under anhydrous conditions.
• Choose a reliable peptide vendor with a proven track record of producing high-quality peptides.