Why Need N-Terminal Sequencing?
Protein N-terminal sequencing remains the method of choice for validating the N-terminal boundaries of recombinant proteins, identifying the N-terminal end of protease resistance structural domains, and identifying proteins isolated from species whose majority of genomes have not been sequenced. Meanwhile, ICH Q6B also requires N-terminal sequence confirmation for drugs. Compared with the Edman degradation method, in MS-based protein N-terminal sequencing, the N-terminal amino acids don't need to be repeatedly cleaved, and the proteins are directly pretreated by digestive enzymes into small fragments to analyze by mass spectrometry. Different peptides can generate fragmentions with different m/z and different spectra to obtain more N-terminal sequence information due to the different masses of most amino acid residues. In addition, mass spectrometry N-terminal sequencing is not affected by the N-terminal end of the protein, and which can also sequence N-terminal blocked and chemically modified amino acids.
Fig. 1. Identification and sequencing of N-terminal peptides in proteins by LC-fluorescence-MS/MS (Malgorzata Monika Vecchi, et al., 2019)
N-Terminal Sequencing Services Offered by Creative Proteomics
Mass Spectrometry-Based N-Terminal Sequencing Services
In the domain of proteomics, the N-terminus of a protein significantly influences its synthesis, structural configuration, and functional attributes. Utilizing the advanced capabilities of our mass spectrometry infrastructure, the team at Creative Proteomics has innovated a specialized approach for the precise sequencing of the N-terminal region of proteins. This methodology is adept at overcoming challenges associated with the analysis of proteins that exhibit N-terminal modifications or blockages, facilitating an in-depth characterization of proteins in their native or modified states.
We provide mass spectrometry-based protein N-terminal sequencing, which avoids the interference of N-terminus closure or modification sites and can achieve 100% coverage of the measured target protein sequence. For protein N-terminal sequencing, we developed a customized sequencing workflow.
Workflow for N-Terminal Sequencing by Mass Spectrometry
We also offer Edman degradation N-terminal sequencing services. Our Edman degradation service employs the classical technique of sequentially removing and identifying amino acids from the N-terminal end of peptides and proteins. This method is highly precise and remains the gold standard for N-terminal sequencing, especially suitable for:
- Confirming the identity of synthesized or purified peptides.
- Verifying protein termini and assessing protein maturation.
- Characterizing protein variants and isoforms.
Fig. 2. Imaged N-terminal sequencing workflow.
Advantages of N-Terminal Sequencing by Mass Spectrometry
The peptide coverage is as high as 100%, and up to 70 amino acid sequences at the N-terminal of the protein can be determined.
Automatic sequence analysis is more accurate.
only need 1-10 μg protein sample can be detected with high sensitivity.
Sequencing is not affected by N-terminal modifications such as N-terminal blocking, PEG and glycosylation.
Sample Requirements for N-Terminal Sequencing by Mass Spectrometry
Sample Type and Quantity
- Amount: 5-10 micrograms (µg) of the protein of interest.
- Purity: Greater than 95% purity is recommended to ensure accurate sequencing.
Sample Preparation
- Buffer Conditions: Avoid buffers with detergents or high salt concentrations. If present, desalting or buffer exchange may be necessary prior to analysis.
- Solvents: Water or volatile solvents (e.g., acetonitrile) are preferred. Non-volatile solvents should be avoided.
- Additives: Avoid additives that could interfere with mass spectrometry, such as glycerol, EDTA, or heavy metals.
Container
- Type: Protein samples should be submitted in low-binding microcentrifuge tubes.
- Labeling: Clearly label each sample with a unique identifier.
Storage and Shipping
- Temperature: Freeze samples at -20°C or -80°C for storage. Ship samples on dry ice to minimize degradation during transit.
- Packaging: Use sturdy shipping containers designed for cryogenic shipment.
Applications of N-Terminal Sequencing
Protein Characterization
- Sequence Confirmation: N-terminal sequencing verifies the primary structure of proteins, confirming the amino acid sequence at the beginning of the protein chain.
- Protein Identification: Determining the N-terminal sequence aids in the identification and classification of proteins, enabling researchers to distinguish between different protein isoforms and variants.
Quality Control in Biopharmaceuticals
- Batch-to-Batch Consistency: N-terminal sequencing ensures consistency in the production of biopharmaceuticals by confirming the integrity of the protein's N-terminus across different batches.
- Identity Testing: It serves as a regulatory requirement for quality control, ensuring that the correct protein sequence is present in the final product.
Post-translational Modification Analysis
- Identification of Modifications: N-terminal sequencing helps identify and characterize post-translational modifications (PTMs) occurring at the N-terminus, such as acetylation, methylation, and cyclization.
- Functional Implications: Understanding N-terminal modifications provides insights into protein function, stability, and localization within the cell.
Protein Engineering and Design
- Site-Directed Mutagenesis: N-terminal sequencing aids in the design and engineering of proteins by confirming the successful introduction or deletion of specific amino acids at the N-terminus.
- Structural Studies: It facilitates the design of protein variants with altered N-terminal sequences to investigate their impact on protein structure and function.
Biomarker Discovery
- Diagnostic Applications: N-terminal sequencing helps identify novel protein biomarkers associated with disease states, aiding in the development of diagnostic assays for early disease detection and monitoring.
- Prognostic Markers: It enables the identification of N-terminal sequences associated with disease prognosis, providing valuable prognostic information for patient management and treatment decisions.
Protein-Protein Interactions
- Mapping Interaction Sites: N-terminal sequencing can elucidate the binding interfaces between proteins, facilitating the study of protein-protein interactions and signaling pathways.
- Drug Discovery: It aids in the identification of small molecule inhibitors or peptides that disrupt protein-protein interactions implicated in disease pathogenesis, offering potential targets for drug development.
Structural Biology and Protein Folding
- Folding Studies: N-terminal sequencing provides insights into the early stages of protein folding and assembly, helping elucidate the mechanisms underlying protein folding kinetics and stability.
- Structure-Function Relationships: It aids in correlating protein structure with function, guiding the design of experiments to probe protein folding pathways and stability.
Case: Analysis of MeCP2 Protein Dynamics and Degradation Rates in Living Cells
Background
Understanding the dynamics and degradation rates of MeCP2 protein variants in living cells is crucial for elucidating their roles in cellular processes and disease pathology. MeCP2 plays a vital role in transcriptional regulation and chromatin organization, and mutations in this protein are associated with neurodevelopmental disorders such as Rett syndrome.
Samples
The study utilized HEK293T and C2C12 cell lines transfected with wild-type (WT) and Ala2Val mutant MeCP2 constructs. Affinity-purified MeCP2 proteins were analyzed using gel-free purification and mass spectrometry techniques.
Technical Methods
Cloning, Mutagenesis, Cell Culture, and Transfections:
- MeCP2_E1 constructs were prepared and transfected into C2C12 and HEK293T cells.
- The Ala2Val mutation was introduced using PCR-based site-directed mutagenesis.
Cell Harvesting and Protein Extraction:
- Cells were harvested, washed, and lysed in lysis buffer.
- Protein concentrations were quantified using a BCA protein assay kit.
Gel-Free Affinity Purification/Immunoprecipitation and Trypsin Digestion for Mass Spectrometry:
- MeCP2 proteins were purified using GFP-Trap® and analyzed by western blotting.
- Tryptic peptides were prepared for mass spectrometry analysis.
Mass Spectrometry to Determine MeCP2 PTMs:
Tryptic peptides were analyzed using a Q-Exactive™ Hybrid Quadrupole-Orbitrap™ mass spectrometer.
Cell Fixation and Confocal Imaging:
Fixed cells were stained with DAPI and imaged using confocal microscopy to study protein localization.
Fluorescence Recovery After Photobleaching (FRAP):
- FRAP assays were conducted to investigate protein recovery and dynamics in living cells.
- Confocal time-lapse imaging was performed pre- and post-bleaching.
Cell Viability Test:
SK-N-SH cells were treated with cycloheximide (CHX) and imaged to assess cell viability.
Cycloheximide Chase Assay:
Neuroblastoma cells were transfected with MeCP2 constructs and treated with CHX for western blot analysis.
Real-Time Bleach-Chase Assays:
HEK293T cells were transfected with MeCP2 constructs and subjected to bleach-chase experiments to study protein degradation rates.
Data Analysis:
Various software tools were employed for data analysis, including Olympus FluoView, MATLAB-based easyFRAP, Microsoft Excel, and GraphPadTM.
Mass spectrometry sequencing of the N-terminal of the MeCP2 protein (in vitro).
Results
The study revealed differences in the mobility, degradation rates, and half-life of WT and Ala2Val mutant MeCP2 proteins in living cells. The Ala2Val mutation led to slower recovery rates and higher degradation rates compared to WT MeCP2, suggesting potential implications for disease pathology. Additionally, no significant differences were observed in protein-chromocenter localization or overall chromatin organization between WT and mutant MeCP2 constructs. These findings provide insights into the functional consequences of MeCP2 mutations and their relevance to neurodevelopmental disorders.
Reference
- Sheikh, Taimoor I., et al. "MeCP2_E1 N-terminal modifications affect its degradation rate and are disrupted by the Ala2Val Rett mutation." Human molecular genetics 26.21 (2017): 4132-4141.
