Sequencing Peptides: Methods for Cutting-Edge Analysis

Peptide sequencing is essential in biochemistry and molecular biology for identifying protein structures, studying post-translational modifications, and developing targeted therapeutics. Advances in analytical techniques have greatly improved the accuracy and efficiency of determining amino acid sequences.

Several methods are commonly used, each with distinct advantages depending on sample complexity and research objectives.

Edman Degradation

Edman degradation is a classical method for sequencing peptides by selectively removing and identifying amino acids from the N-terminus of a polypeptide chain. Developed by Pehr Edman in the 1950s, this technique remains relevant for determining short peptide sequences with high precision. The process relies on phenyl isothiocyanate (PITC), which reacts with the free amino group of the terminal residue under mildly alkaline conditions to form a phenylthiocarbamoyl derivative. Subsequent acid treatment cleaves this modified residue, generating a cyclic phenylthiohydantoin (PTH) derivative that can be identified using high-performance liquid chromatography (HPLC).

The specificity of Edman degradation allows for sequential identification of amino acids without disrupting the rest of the peptide chain. However, efficiency decreases with longer sequences due to incomplete cleavage and side reactions, typically limiting its application to peptides of around 30–50 residues. Blocked N-termini or post-translational modifications can also interfere, requiring additional sample preparation. Despite these limitations, Edman degradation remains valuable for analyzing purified peptides, particularly when mass spectrometry is not feasible or when confirming sequence data.

This method has been instrumental in characterizing bioactive peptides, such as hormones and antimicrobial peptides. For example, sequencing insulin’s A and B chains provided critical insights into its structure and function. Researchers also use Edman degradation to verify recombinant protein sequences in biopharmaceutical production. While newer technologies have expanded sequencing capabilities, this method continues to serve as a reliable approach for targeted peptide analysis.

Protease Cleavage Methods

Protease cleavage fragments peptides and proteins into smaller segments for sequencing and structural analysis. Proteolytic enzymes generate predictable peptide fragments based on amino acid recognition motifs. Trypsin, for example, hydrolyzes peptide bonds at the carboxyl side of lysine and arginine residues, producing well-defined fragments useful in mass spectrometry. Other enzymes, such as chymotrypsin and pepsin, target hydrophobic and aromatic residues, offering alternative cleavage patterns.

Controlled digestion with proteases enables sequence reconstruction through overlapping fragment analysis. This is particularly useful for large or complex proteins that are difficult to sequence directly. Using multiple proteases with distinct cleavage specificities increases sequence coverage and resolves ambiguities. For instance, combining trypsin with Glu-C, which cleaves at the C-terminal side of glutamic and aspartic acid residues, provides additional sequence information. This approach is also valuable for mapping post-translational modifications, pinpointing modification sites within a protein sequence.

In addition to enzymatic digestion, chemical cleavage methods offer an alternative for fragmenting peptides. Cyanogen bromide selectively cleaves at methionine residues, creating distinct peptide fragments that complement enzymatic digestion. Hydroxylamine can cleave asparagine-glycine bonds, while formic acid hydrolyzes peptide linkages at aspartic acid residues under controlled conditions. These chemical approaches are useful when proteins resist enzymatic digestion or when specific cleavage sites are needed for structural mapping.

Mass Spectrometry Approaches

Mass spectrometry has revolutionized peptide sequencing by offering unparalleled sensitivity, speed, and accuracy. Unlike traditional methods that rely on stepwise degradation or enzymatic digestion alone, mass spectrometry directly analyzes peptide masses and fragmentation patterns, making it particularly useful for complex mixtures and modified peptides. Modern mass spectrometers, such as quadrupole time-of-flight (QTOF) and orbitrap instruments, provide high-resolution data that enable precise mass determination down to fractions of a Dalton.

A central technique in peptide sequencing via mass spectrometry is tandem mass spectrometry (MS/MS), which involves multiple stages of ion fragmentation and analysis. Peptides are first ionized using electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI), generating charged species that can be further fragmented in a collision-induced dissociation (CID) or higher-energy collisional dissociation (HCD) process. These fragmentation events break peptide bonds in predictable ways, typically generating b- and y-ion series that reflect the sequence of amino acids. By analyzing these ion series, researchers can reconstruct the peptide’s primary structure with high confidence, even in complex biological samples.

Advanced techniques such as electron-transfer dissociation (ETD) and electron-capture dissociation (ECD) have expanded mass spectrometry-based sequencing. Unlike CID and HCD, which primarily fragment peptide backbones, ETD and ECD preserve labile post-translational modifications while cleaving along the peptide chain. This makes them particularly valuable for studying phosphorylation, glycosylation, and disulfide bonding patterns, which play significant roles in protein function. The integration of these fragmentation techniques into hybrid mass spectrometers has enhanced peptide analysis, enabling researchers to characterize intricate structural features with greater accuracy.