Can’t Measure Protein N-Terminus? 2 Common Issues + Solutions, Save This!

In proteomics research,N-terminal sequencingis of significant importance for elucidating biological issues such as protein start sites, post-translational modifications (PTMs), signal peptide cleavage, and degradation pathways. However, many researchers often encounter a frustrating phenomenon in practice:The N-terminus of the protein simply cannot be detected!

 

This article will analyze two common technical bottlenecks from a mechanistic perspective and provide practical solutions to help you improve the detection rate of N-termini. If you're working on projects related to protein modifications, protein degradation, or antibody validation, this article is worth saving.

 

Issue 1: Protein N-terminus is blocked, mass spectrometry cannot identify it

1. Underlying reason: Post-translational modification masks the N-terminus

In eukaryotic cells, after protein translation is complete, the N-terminus often undergoes a series of post-translational modifications, such as:

（1）N-terminal acetylation

(2) Formylation

(3) Signal peptide or initial methionine (Met) cleavage

 

These modifications can inactivate the free amino group at the N-terminus, affecting Edman degradation or mass spectrometry's ability to recognize the true N-terminus, leading to signal loss or misinterpretation.

 

2. Solution: Targeted N-terminal enrichment technology

To improve detection sensitivity, the current mainstream approach is to usespecific N-terminal labeling and enrichment strategiessuch as:

（1）TAILS（Terminal Amine Isotopic Labeling of Substrates）

（2）COFRADIC（Combined Fractional Diagonal Chromatography）

(3) Specialized N-terminomics kits or tagging technologies

 

These methods selectively label and enrich the true N-terminus by blocking internal peptide amino groups, significantly enhancing detection signal and accuracy. At Biotech Pack, we have established a matureTAILS + high-resolution mass spectrometryintegrated platform, suitable for various sample types including mammals, yeast, and plants, helping customers efficiently analyze protein N-terminal structure and modification states.

 

Issue 2: Protease cleavage sites are close to the N-terminus, resulting in loss of N-terminal peptide

1. Underlying reason: Improper enzyme cleavage strategy design

Most proteomics experiments use trypsin for protein digestion. However, trypsin preferentially cleaves at K/R sites, and the N-terminus of proteins often lacks ideal cleavage conditions, easily causing:

（1）N-terminal peptide being too short (<6 aa), not recognized by mass spectrometry

(2) or N-terminal peptide lacking charged groups, resulting in low ionization efficiency

 

This is one of the fundamental reasons for the 'silence' of N-terminal signals in many samples.

 

2. Solution: Multi-enzyme combined cleavage strategy

To improve N-terminal detection efficiency, one can use:

（1）Lys-C, Asp-N, Glu-C and other specific proteases

（2）dual-enzyme or sequential enzymatic digestion (e.g., Lys-C + Trypsin)

（3）adjust the pH of the digestion buffer to optimize exposure of cleavage sites

 

In addition, pairing withhigh-sensitivity LC-MS/MS systemsandoptimized high-resolution mass spectrometry scanning parameterscan also significantly enhance the detection capabilities of N-terminal peptides.Biotech Packoffers flexible multi-enzyme digestion solutions and combines advanced platforms such as Orbitrap Exploris 480 for targeted N-terminal detection, providing robust data support for antibody drug characterization, protein degradation target screening, and other applications.

 

Summary: Incomplete detection of protein N-termini is both a technical blind spot and an optimization breakthrough

 

Common Issues	Technical Barriers	Solution Strategies
N-terminal blocked by post-translational modifications	Difficult to detect directly	N-terminal enrichment labeling method (e.g., TAILS)
Improper design of cleavage sites	Peptides too short/lack charged groups	Multi-enzyme combination cleavage + high-sensitivity mass spectrometry

 

With the rise of post-translational modification research and targeted degradation drug development, precise N-terminal identification will become increasingly important. If you are planning related projects, feel free to contact Biotech Pack. We can customize the most suitable N-terminal sequence research plan for you, providing full-process support from sample preparation to data analysis. Biotech Pack offers protein N-terminal sequence analysis services. Our 'one-stop' service can save you time and effort, helping you conduct related research more efficiently.

 

Biotech Pack--Characterization of biological products, high-quality service provider of multi-omics biomass spectrometry detection

 

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