What is Edman Degradation Sequencing?
Edman degradation sequencing, allows for the tagging and cleavage of peptides from the N-terminus without breaking peptide bonds between other amino acid, is a mature protein sequencing method. With the development of mass spectrometry technology, the use of Edman degradation sequencing began to decrease, but Edman degradation sequencing remains a powerful and irreplaceable method for protein N-terminal sequencing. As an established sequencing method, Edman Degradation sequencing provides more accurate protein sequence data than MS. Which Could be used to verify the N-terminal boundaries of recombinant proteins or to determine the N-terminus of protease resistance domains, especially when the protein or domain is >40 - 80 kda or is not easily purified. In addition, some new proteins and peptides for which sequence databases are not available for MS/MS database searches can be analyzed using Edman degradation.
Fig. 1. The Edman degradation reactions and conversion step. (Khalid Z M, et al., 2021)
Edman N-terminal Sequencing in Creative Proteomics
Creative Proteomics relies on the company's existing Edman sequencing system to provide N-terminal sequencing services for purified protein products, antibodies and protein vaccines for scientific researchers and scientific research customers. Using our sequencing system, the sequence information of the N-terminal 30 amino acids can be determined, while using a specific protein loading system, the N-terminal 60-70 amino acids can be determined. In addition, our professional protein sequencing platform also provides an N-terminal sequence analysis platform based on mass spectrometry, which can detect blocked and modified protein ends. The advantages of the two complement each other to ensure the smooth progress of N-terminal sequencing. The Edman based protein sequencing we provide is also suitable for the folowing services.
In order for you have a clearer understanding about our Edman sequencing services, Creative Proteomics has customized the following complete sequencing process for your protein.
Ⅰ. Under alkaline conditions, PITC binds to the free amino group at the N-terminus of the protein.
Ⅱ. In acidic solution, the N-terminal residue is cleaved.
Ⅲ. The PITC-bound residues were converted into more stable PTH residues.
Ⅳ. The amino acid species was determined by on-line HPLC analysis according to the elution time.
Sample Requirements of Edman Sequencing
1. Liquid Samples
- Amount: 1-10 μg.
- Purity: >90%.
- Avoid using Tris, glycine, guanidine, glycerol, sucrose, SDS, Triton, X-100, Tween, and ammonium sulfate in the buffer.
2. Electroblotted Samples
- Protein samples are separated by SDS-PAGE to ensure that the purity of the samples meet the requirements of sequencing.
- The protein samples on SDS-PAGE were transferred to PVDF membranes, (Note: PVDF membrane can be stained with Coomassie blue or Poncaue red not recommended with Sliver stain), followed by washing with ultra-pure water. The washing steps must be repeated several times, when glycine-buffer is used bmitting samples.
- The target protein band on the obtained PVDF membrane were cut off with a clean scalpel.
Our Technical Advantages
- N-terminal sequencing of most proteins can be determined, with complementary mass spectrometry platform.
- The measured N-terminal amino acids are as high as 60-70.
Applications
Creative Proteomics provides comprehensive Edman degradation sequencing projects for global customers. But if you have any special requirement, you are welcome to contact us for custom service. We look forward to your happy cooperation.
Question: Is there a requirement for protein sample purity in N-terminal sequencing?
Answer: Yes, ideally, the purity should be above 95%; otherwise, with multiple amino acids eluting in each cycle, it becomes challenging to attribute them to the protein sequence.
Question: If a protein has two or three chains, how can N-terminal sequencing be conducted?
Answer: Firstly, perform SDS-PAGE electrophoresis to separate the bands. Then transfer the protein from the gel to a PVDF membrane. After Coomassie Brilliant Blue staining, cut the corresponding bands for sequencing.
Two points to note: Avoid using Tris-glycine buffer during transfer, as it can cause higher background in N-terminal sequencing. CAPS buffer is recommended. Also, when staining the PVDF membrane, use Ponceau S instead of Coomassie Brilliant Blue.
Question: What kind of samples are not suitable for N-terminal sequencing using the Edman degradation method to analyze the sequence?
Answer: First, samples with blocked N-termini, as the reaction cannot proceed when the α-amino group at the N-terminus is modified. Second, samples with too many non-standard amino acids in the targeted N-terminal sequence. In such cases, where there are no corresponding standards, N-terminal sequencing is not feasible. Mass spectrometry can be used for sequence confirmation in these situations.
Case: Identification and Characterization of a Novel Muscle-Type nAChR Inhibitory Peptide, Macoluxin, from the Venom of the Cat-Eyed Snake M. colubrinus
Background
The study focuses on exploring the venom of the rear-fanged cat-eyed snake M. colubrinus, aiming to identify and characterize a peptide with inhibitory effects on muscle-type nicotinic acetylcholine receptors (nAChRs). This research contributes to understanding the diversity of snake venom components and their potential as sources for bioactive peptides.
Samples
Venom extracted from M. colubrinus specimens kept in the serpentarium.
Membranes of Torpedo californica used for competitive binding experiments.
ALEXA 488-labeled α-Bgt employed as a marker for nAChR binding.
Technical Methods
1. Venom Extraction and Peptide Isolation:
- Venom dissolved in 0.1 M ammonium acetate.
- Superdex 75 column used for initial separation.
- Further purification via reversed-phase chromatography on Jupiter C18 columns.
2. Amino Acid Sequencing:
- Primary structure determined by Edman degradation using a PPSQ-33A sequencer.
3. Peptide Synthesis:
- Macoluxin synthesized through solid-phase peptide synthesis.
4. Biological Activity of Macoluxin:
- Competitive Fluorescence Analysis:
- 96-well plate used for fluorescence assays.
- Binding buffer containing NaCl, KCl, KH2PO4, Na2HPO4, Tween-20, pH 7.4.
- ALEXA488-Bgt employed for fluorescence measurement.
- Nonspecific binding determined with excess α-cobratoxin.
- Cell Cultivation and Transient Transfection:
- Neuro2a and HEK293 cells used for transfection.
- Lipofectamine 2000 and plasmids encoding muscle-type nAChR subunits utilized.
- Detection of Changes in Intracellular Calcium:
- Genetically encoded Case12 sensor employed for calcium level determination.
- Real-time imaging with an Olympus XM-10 CCD camera.
- Competitive Radioligand Binding Assay:
- Competitive binding experiments with membranes of T. californica electric organ or GH4C1 cells.
- [125I]-labeled α-Bgt used for radioactive binding.
- Electrophysiological Measurements:
- Xenopus oocytes used for electrophysiological measurements.
- Macoluxin preincubated with oocytes before coapplication with acetylcholine.
- Reversibility of nAChR inhibition tested by washing the receptor.
Results
The venom of M. colubrinus inhibits nAChR, marking the first identification of such activity in a rear-fanged snake.
Macoluxin, a peptide from M. colubrinus venom, competitively inhibits muscle-type nAChR.
Amino acid sequence analysis reveals macoluxin's high similarity to a fragment of snake venom metalloproteinases.
Macoluxin's inhibitory effect on nAChR is demonstrated through competitive binding assays and electrophysiological experiments.
The study suggests that venom proteins can serve as precursors for diverse peptide toxins with distinct biological activities.
The isolation of active compound from the venom of M. colubrinus
The separation of fraction 18 by reversed-phase chromatography on a Jupiter C18 column (4. 6 × 250 mm) in an acetonitrile concentration gradient of 20–30% for 60 min at a flow rate of 1 mL/min. The horizontal line indicates the active fraction.
Reference
- Kryukova, E. V., et al. "A New Peptide from the Venom of the Madagascar Cat-Eyed Snake Madagascarophis colubrinus Blocks Nicotinic Acetylcholine Receptor." Russian Journal of Bioorganic Chemistry 49.3 (2023): 529-537.
