Protein De Novo Sequencing: 10 Key Challenges and How to Overcome Them
Protein de novo sequencing has shown distinct value in determining unknown amino acid sequences. However, the process still faces several significant challenges, spanning experimental design, data analysis, and biological complexity. These challenges require technological innovations and interdisciplinary approaches to overcome. This paper explores solutions for 5 key issues, integrating the latest advancements in technology.
Protein De Novo Sequencing Challenge 1: Incomplete Fragment Ions and Spectral Noise
1. Problem Essence
During peptide fragmentation in mass spectrometry, incomplete ion coverage can occur due to fragmentation biases or uneven energy distribution. This may lead to missing key fragment ions, such as only generating b ions and not y ions. Additionally, noise introduced by sample impurities or ionization competition-where multiple molecules compete for ionization-can interfere with the accurate analysis of the target peptides.
2. Solutions
(1) By combining multiple fragmentation techniques, such as Collision-Induced Dissociation (CID), Electron Transfer Dissociation (ETD), and Ultraviolet Photodissociation (UVPD), it is possible to generate a wider variety of fragment ions, thereby improving ion coverage.
(2) The use of AI-driven spectral denoising algorithms, particularly deep learning models, can predict theoretical spectra, helping to identify and eliminate noise, thus improving the signal-to-noise ratio and facilitating more accurate analysis.
Protein De Novo Sequencing Challenge 2: Ambiguity of Isomeric Amino Acids with Identical Mass
1. Problem Essence
Some amino acids, such as leucine and isoleucine, or lysine and glutamine, have extremely close molecular masses, making them difficult to distinguish using traditional mass spectrometry techniques. This leads to ambiguity in sequence interpretation, as these subtle differences in mass can significantly affect the accuracy of the determined sequence.
2. Solutions
(1) Ion Mobility Spectrometry (IMS) can be used to measure the spatial differences in the structure of ions, enhancing the ability to differentiate isomeric amino acids with identical masses. This method provides additional structural information that is not captured by traditional mass spectrometry alone.
(2) Special fragmentation techniques such as Electron-Activated Dissociation (EAD) can help distinguish amino acid isomers by exploiting their distinct ion characteristics, providing an alternative to traditional fragmentation methods.
Protein De Novo Sequencing Challenge 3: Interference from Post-translational Modifications (PTM)
1. Nature of the Problem
Phosphorylation, glycosylation, and other post-translational modifications alter the mass of peptides. When the type and site of modification are unknown, traditional database search methods face significant challenges in covering all possible modifications and their variants.
2. Solution
(1) Employ open modification search algorithms that allow for a broad range of mass shifts, thus enabling the identification of unknown modifications.
(2) Use multi-stage mass spectrometry techniques (e.g., MS³) to further fragment modified peptides and acquire detailed information on the modification sites.
Protein De Novo Sequencing Challenge 4: Difficulty in Long Peptide and Full Protein Sequencing
1. Nature of the Problem
For longer peptides and entire proteins, low ionization efficiency combined with the complexity of fragment spectra results in decreased sequence coverage, ultimately affecting the completeness of the analysis.
2. Solution
(1) Apply top-down mass spectrometry strategies in conjunction with high-resolution mass spectrometers to analyze full protein fragments.
(2) Utilize charge-reduction techniques to decrease the charge state, simplify spectra, and enhance the efficiency of long peptide and full protein analysis.
Protein De Novo Sequencing Challenge 5: Insufficient Sensitivity for Low-abundance Protein Detection
1. Nature of the Problem
In complex biological samples, high-abundance proteins dominate the signal, which interferes with the detection of low-abundance target proteins, thereby compromising the sensitivity of de novo protein sequencing.
2. Solution
(1) Use microfluidic chips and other sample preparation techniques to enrich target proteins, thus increasing the relative abundance of low-abundance proteins.
(2) Employ novel ionization-enhancing materials, such as nanostructured substrates, to improve ionization efficiency and increase signal intensity.
The 5 major challenges in protein de novo sequencing reflect the ongoing struggle between technical limitations and biological complexity. Current solutions are showing a clear trend toward multi-technology integration: innovations in high-precision mass spectrometry hardware, optimization of artificial intelligence algorithms, and breakthroughs in single-molecule sequencing are collectively pushing the boundaries of technology. In the next decade, as clinical applications and industrial integrations evolve, this technology is likely to transition from being a “research tool” to becoming a core infrastructure within the life sciences sector. MtoZ Biolabs, a professional provider of high-quality multi-omics services in biological mass spectrometry, offers de novo protein sequencing services to clients.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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