MS-Based N-Terminal Sequencing: Sample Preparation and Data Analysis
N-terminal sequencing is a critical approach in proteomics for determining the N-terminal amino acid sequence of proteins and peptides. It plays an essential role in investigating post-translational modifications, protein maturation, degradation pathways, and functional mechanisms. Compared to the traditional Edman degradation method, MS-based N-terminal sequencing has emerged as a powerful analytical tool due to its high sensitivity, high throughput, and compatibility with complex biological samples.
This review systematically outlines the workflow of MS-based N-terminal sequencing, focusing on three key aspects: sample preparation, mass spectrometry analysis, and data interpretation. Additionally, it highlights critical technical considerations in these processes.
Sample Preparation: Efficient Enrichment of N-Terminal Peptides
Sample preparation is the foundational step for achieving efficient N-terminal sequencing. To enhance the sensitivity and specificity of MS-based detection, several strategies are employed to reduce sample complexity and selectively enrich N-terminal peptides. A typical workflow includes protein denaturation, reduction, and alkylation, which help eliminate disulfide bond interference and improve enzymatic digestion efficiency.
Proteolysis using specific enzymes such as trypsin, GluC, and AspN is commonly performed to generate defined peptide fragments. However, conventional enzymatic digestion often yields a high proportion of non-N-terminal peptides. To address this issue, selective enrichment techniques, including chemical labeling and solid-phase capture, are applied to isolate true N-terminal peptides. For instance, formaldehyde or acetone-based chemical derivatization can label protein N-termini, while solid-phase affinity-based approaches can selectively capture N-terminal peptides, thereby minimizing background interference. Furthermore, for specific sample types, nonspecific proteases (e.g., proteinase K) or limited proteolysis strategies are employed to generate N-terminal peptides suitable for sequencing.
Mass Spectrometry Analysis: Precise Characterization of N-Terminal Sequences
Mass spectrometry analysis is the core technological component of N-terminal sequencing. The combination of liquid chromatography-mass spectrometry (LC-MS/MS) with high-resolution platforms such as Orbitrap and TOF-MS enables precise characterization of N-terminal peptides in complex biological samples.
The choice of fragmentation technique significantly impacts the quality of N-terminal sequence data. Collision-induced dissociation (CID), a widely used fragmentation method, generates abundant b- and y-ions that facilitate peptide sequencing. Meanwhile, advanced fragmentation techniques such as higher-energy collision dissociation (HCD) and electron transfer dissociation (ETD) provide superior fragmentation patterns for N-terminal peptides carrying post-translational modifications (e.g., phosphorylation, glycosylation). To further improve sequence coverage, both data-dependent acquisition (DDA) and data-independent acquisition (DIA) strategies are commonly employed, ensuring comprehensive identification and characterization of N-terminal peptides across diverse sample types.
Data Analysis: Accurate Identification of N-Terminal Sequences
Data analysis is a crucial step in N-terminal sequencing. First, after preprocessing the raw mass spectrometry data, which includes peak extraction and mass-to-charge ratio calibration, peptide matching is performed. Traditional database search methods (such as SEQUEST, Mascot, and MaxQuant) are commonly used for N-terminal peptide identification, but they are often integrated with open search or blind search strategies to account for potential N-terminal modifications.
In recent years, spectral library-based search methods (such as SpectraST) have proven effective in N-terminal sequencing, particularly in analyzing complex protein modifications. Furthermore, the incorporation of deconvolution algorithms and machine learning approaches has facilitated automated extraction of N-terminal peptide information from mass spectrometry data, greatly enhancing analytical efficiency.
Mass spectrometry-based N-terminal sequencing has wide-ranging applications in protein identification, post-translational modification analysis, and protein processing research. The advancement of this field is driven by efficient sample preparation strategies, optimized mass spectrometry techniques, and state-of-the-art data processing methodologies. MtoZ Biolabs offers high-quality mass spectrometry-based N-terminal sequence analysis services. For further inquiries, please contact us.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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