Protein Full-Length Sequencing: Complete Sequence Coverage Scheme with High Precision and Low Sample Size

In life sciences and biopharmaceutical development, the structural and functional characteristics of proteins are intrinsically linked. Comprehensive knowledge of a protein’s full-length sequence is fundamental for understanding its biological functions, identifying mutation sites, and characterizing post-translational modifications. Conventional mass spectrometry-based approaches typically rely on enzymatic digestion, fragmenting proteins into peptides for identification, which often results in partial sequence coverage. Protein full-length sequencing integrates multi-enzyme strategies with high-resolution mass spectrometry to recover complete protein sequences. As an emerging complementary approach, FLPS is gaining increasing attention in proteomics.

 

What Is Protein Full-Length Sequencing and Why Is It Important?

Protein full-length sequencing aims to determine the entire amino acid sequence of a protein, including the N-terminal, C-terminal, and internal regions, thereby overcoming the limitations of traditional peptide-based methods that may miss critical domains or modification sites. This technique offers unique advantages in several key applications:

1. Antibody sequence deconvolution and patent submission: Enables precise identification of full-length light and heavy chain sequences, supporting antibody humanization and intellectual property claims.

2. Biopharmaceutical quality assurance: Facilitates the detection of sequence variants such as mutations, isoforms, and splice variants, ensuring product consistency.

3. Functional characterization of novel proteins: Aids in the discovery of unconventional translational isoforms, uncharacterized modification patterns, and retained signal peptides in endogenous proteins.

 

Technical Challenges: Why Is Full-Length Sequencing Difficult to Achieve?

Unlike genome sequencing, protein full-length sequencing presents several inherent challenges:

1. Limited peptide coverage: Standard proteolytic digestion may not generate overlapping peptides sufficient to reconstruct the full-length protein sequence.

2. Complexity of post-translational modifications (PTMs): PTMs such as phosphorylation and glycosylation can alter fragmentation behavior and hinder sequence reconstruction.

3. Presence of isomeric forms: Coexisting structural isomers, including isobaric peptides, complicate database-dependent identifications and contribute to sequence ambiguity.

 

Protein Full-Length Sequencing Strategy: High-Precision Workflow Combining Multi-Enzyme Digestion and Tandem Mass Spectrometry

To overcome the aforementioned challenges, MtoZ Biolabs has developed a high-coverage protein full-length sequencing workflow that integrates multiple enzymatic digestion strategies, multi-stage tandem mass spectrometry (MSⁿ) acquisition, and dual-mode de novo/database-based interpretation algorithms:

1. Multi-Enzyme Digestion Combined with Diverse Preparation Strategies

We employ a combination of proteases including trypsin, chymotrypsin, and Glu-C to increase sequence coverage by overcoming structural domain constraints. Additionally, non-enzymatic approaches such as top-down and middle-down sequencing are supported to retain native post-translational modifications (PTMs).

 

2. High-Resolution Tandem Mass Spectrometry Instrumentation

Utilizing advanced instruments such as Orbitrap Eclipse and timsTOF, alongside dual fragmentation techniques (HCD and ETD), enables enhanced identification of PTMs and improves the detection sensitivity of low-abundance peptides.

 

3. Integrated De Novo and Database-Driven Analysis

By integrating de novo sequencing with database searches through cross-validation, our approach ensures accurate and comprehensive identification of novel mutations and previously uncharacterized protein sequences.

 

4. Minimal Sample Input with Broad Sample Compatibility

MtoZ Biolabs’ Protein Full-Length Sequencing service supports workflows starting from as little as 1 µg of protein and is compatible with a wide array of sample types, including serum, cell lysates, purified proteins, and tissue homogenates—making it particularly advantageous for clinical and limited-sample studies.

 

Summary: Advancing from Peptide Fragments to Complete Protein Sequences

Protein Full-Length Sequencing is rapidly evolving from exploratory research to a standardized, high-throughput solution characterized by strong reproducibility. This technology extends the frontiers of proteomics and provides critical sequence-level insights for antibody drug development, biopharmaceutical quality control, and basic life sciences research. At MtoZ Biolabs, we are committed to delivering high-quality, full-length sequence data tailored to your research needs, enabling in-depth analysis of protein function down to each individual amino acid residue.

 

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

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Protein Full-Length Sequencing Service