De Novo Protein Sequencing: 5 Major Challenges and Solutions

De Novo Protein Sequencing demonstrates unique value in resolving unknown amino acid sequences, but its technical processes still face multiple challenges. These challenges involve experimental design, data interpretation, and biological complexity, necessitating breakthroughs through technological innovation and interdisciplinary integration. The following discussion addresses solutions to five core issues based on recent technological advancements.

 

1. Challenge 1 in De Novo Protein Sequencing: Incomplete Fragment Ions and Spectrum Noise

1. Nature of the Problem

During peptide fragmentation in mass spectrometry, preference for certain breaks or uneven energy distribution leads to incomplete fragment ion coverage (e.g., only generating b ions while missing y ions). Additionally, noise signals introduced by sample impurities or ionization competition interfere with the analysis of target peptides.

 

2. Solutions

(1) Use multiple fragmentation modes (such as CID, ETD, UVPD) together to supplement different ion types and improve coverage.

(2) Apply AI-based spectrum denoising algorithms, using deep learning models to predict theoretical spectra and assist in noise removal, thereby improving signal-to-noise ratio.

 

2. Challenge 2 in De Novo Protein Sequencing: Ambiguity of Isobaric Amino Acids

1. Nature of the Problem

Some amino acids (such as leucine and isoleucine, lysine and glutamine) have extremely similar masses, making it difficult to distinguish them using traditional mass spectrometry, which affects the accuracy of sequence derivation.

 

2. Solutions

(1) Combine ion mobility spectrometry (IMS) to measure spatial structural differences of ions, enhancing the ability to differentiate isobaric amino acids.

(2) Use special fragmentation methods like electron activated dissociation (EAD) to utilize different ion characteristics for distinguishing amino acid isomers.

 

3. Challenge 3 in De Novo Protein Sequencing: Interference from Post-translational Modifications (PTMs)

1. Nature of the Problem

Modifications like phosphorylation and glycosylation alter peptide mass, and when modification types and sites are unknown, traditional database search methods struggle to cover all possibilities.

 

2. Solutions

(1) Use open modification search algorithms that allow for a wide range of mass shifts to identify unknown modifications.

(2) Employ multi-stage mass spectrometry (such as MS³) to further fragment modified peptides, obtaining detailed information on modification sites.

 

4. Challenge 4 in De Novo Protein Sequencing: Difficulties in Sequencing Long Peptides and Complete Proteins

1. Nature of the Problem

For longer peptides and complete proteins, low ionization efficiency and high fragmentation complexity lead to decreased sequence coverage, affecting overall analysis.

 

2. Solutions

(1) Apply top-down mass spectrometry strategies, combined with high-resolution mass spectrometers, to analyze complete protein fragments.

(2) Use charge reduction techniques to lower charge states, simplifying spectra and improving the efficiency of analyzing long peptides and complete proteins.

 

5. Challenge 5 in De Novo Protein Sequencing: Insufficient Sensitivity for Detecting Low-Abundance Proteins

1. Nature of the Problem

In complex biological samples, high-abundance proteins dominate the signal, masking low-abundance target proteins and affecting the sensitivity of de novo sequencing.

 

2. Solutions

(1) Use pre-treatment methods like microfluidic chips to enrich target proteins, increasing the relative abundance of low-abundance proteins.

(2) Utilize novel ionization-enhancing materials (such as nanostructured substrates) to boost ionization efficiency and enhance signal strength.

 

The essence of the five challenges in De Novo Protein Sequencing is the interplay between technological limits and biological complexity. Current solutions exhibit a trend of multi-technology collaboration: advancements in high-precision mass spectrometry hardware, optimization of AI algorithms, and breakthroughs in single-molecule sequencing collectively push the boundaries of the technology. Over the next decade, as clinical translation and industrial adaptation progress, this technology is expected to evolve beyond its 'research tool' status to become a core infrastructure in the field of life sciences.Bio-Techne BiotechAs a professional provider of high-quality services in biological mass spectrometry and multi-omics, we offer de novo protein sequencing services to our clients.

 

Bio-Techne Biotech -- Characterization of Biological Products, High-Quality Services in Multi-Omics Biological Mass Spectrometry

 

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De Novo Sequencing