Peptide Sequencing Techniques: Challenges and Breakthroughs

What Is Peptide Sequencing? Why is it so Critical?

Peptides are short chains composed of 2–50 amino acid residues, which can be degradation products of proteins, functional signaling molecules, bioactive drugs, or even vaccine antigen epitopes or TCR peptide segments.

The primary objectives of peptide sequencing include:

• Confirming the amino acid sequence of antibody CDR regions

• Identifying endogenous bioactive peptides (such as hormones and cytokines)

• Verifying the structure of degradation products from biopharmaceuticals

• Performing quality control and impurity analysis of peptide drugs

• Conducting epitope screening and studies on T cell recognition sites

Due to the short length of peptides, conventional protein sequencing workflows are often insufficient, prompting the development of specialized sequencing methods tailored to the unique properties of peptides.

 

Mainstream Technical Approaches for Peptide Sequencing

Currently, peptide sequencing techniques can be broadly categorized into two major approaches: mass spectrometry-based sequence analysis (MS/MS) and chemical degradation methods (such as Edman degradation).

1. LC-MS/MS Tandem Mass Spectrometry Sequencing

(1) The core workflow includes:

• Sample purification → Removal of high-background salt ions and impurities

• Ionization (ESI or MALDI) → Converting peptides into charged particles

• Primary mass spectrometry to select precursor ions → Precise selection of target peptides

• Secondary mass spectrometry fragmentation (CID/HCD/ETD) → Generating fragmentation spectra

• Sequence analysis: Reconstruction of sequences through database searches or de novo algorithms

(2) Key advantages:

• Rapid, high-throughput, and high-resolution sequencing

• Capability to integrate identification of modifications and quantitative analysis

 

2. Edman Degradation Method

Edman degradation is one of the most established chemical sequencing techniques, which determines sequences by cyclically removing and identifying N-terminal amino acid residues.

(1) Key advantages:

• High accuracy, particularly suited for pure peptide samples

• Strong analytical performance for short peptides (less than 30 amino acids)

(2) Limitations:

• Inapplicable to peptides with blocked or modified N-termini

• Low throughput and limited automation potential

With advances in mass spectrometry sensitivity and bioinformatics algorithms for sequence reconstruction, modern peptide sequencing is increasingly shifting toward mass spectrometry combined with AI-assisted sequence assembly.

 

Key Challenges in Peptide Sequencing

Although technical methodologies have become increasingly advanced, peptide sequencing still encounters several technical bottlenecks in practice:

1. Complex Modification Types Influencing Fragmentation Patterns

Natural or synthetic peptides often exhibit diverse post-translational modifications (PTMs), including phosphorylation, hydroxyproline, and N-terminal acetylation. These modifications can affect the fragmentation efficiency in mass spectrometry and complicate sequence inference.

 

2. Difficulty in Distinguishing Isobaric Peptides

Peptides contain numerous isobaric species (e.g., Leu/Ile, Gln/Lys) that cannot be directly differentiated by mass spectrometry and necessitate additional chemical validation methods or AI-assisted modeling approaches.

 

3. Weak Signals and High Background Interference

Compared to intact proteins, peptides tend to generate weaker signals, especially in complex biological matrices (e.g., plasma, cell lysates), which substantially increases the complexity of sequencing efforts.

 

4. N-Terminal Modifications and Blocking Issues

Some biological samples contain peptides with blocking modifications that hinder Edman degradation at the N-terminus and compromise the accuracy of database matching algorithms.

 

Technological Breakthroughs and Frontier Developments

To address these challenges, both the scientific and industrial communities have made significant strides in multiple areas:

1. Application of High-Resolution Mass Spectrometry Systems

Advanced platforms such as Orbitrap Eclipse and timsTOF Pro 2 deliver higher-quality MS/MS spectra, enhancing the resolution of isobaric peptides and improving the detection sensitivity for low-abundance signals.

 

2. Multi-Enzyme Digestion and Segmented Synthesis Strategies

By employing multiple specific enzymes (e.g., Trypsin, Glu-C, Chymotrypsin) for complementary cleavage of peptide segments, the sequencing window is broadened, and sequence coverage is maximized.

 

3. AI-Powered De Novo Sequence Reconstruction Algorithms

Deep learning models (e.g., DeepNovo, PointNovo, pNovo+) can reconstruct amino acid sequences from fragmentation patterns, particularly beneficial for sequences absent from databases or for highly modified peptides.

 

4. Integration of Structural Modeling and Modification Site Prediction

By combining AlphaFold-based structural prediction with modification site databases (e.g., Unimod, dbPTM), key functional peptide regions can be identified and structurally verified, thereby enhancing biological interpretability.

 

MtoZ Biolabs Peptide Sequencing Solutions

In research and project development, peptide sequencing demands not only technical precision but also a comprehensive solution from sample preparation to data interpretation. MtoZ Biolabs has established a peptide sequencing service platform tailored for various application scenarios, offering the following core advantages:

• A high-sensitivity platform equipped with high-resolution mass spectrometry systems such as Orbitrap Eclipse and timsTOF

• Customized multi-enzyme digestion strategies to maximize sequence coverage based on peptide segment properties

• AI-assisted analysis engine integrating database matching and de novo prediction to accurately identify unknown peptide sequences

• Robust modification site identification capabilities supporting common PTMs such as phosphorylation, acetylation, and glycosylation

• Comprehensive output reports including sequence spectra, modification annotations, and structural predictions

 

With advancements in mass spectrometry instrumentation and the continual refinement of AI algorithms, peptide sequencing is evolving from mere “sequence acquisition” to comprehensive “functional interpretation”. Future developments in peptide sequencing will not only prioritize accuracy but also emphasize biological insights—including functional domains, modification patterns, structural information, and biological activity. The deep integration of bioinformatics and experimental platforms will further unlock the potential of peptide sequencing in drug discovery, immunotherapy, and mechanistic research.

 

MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.

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