Edman Degradation-based Protein N-terminal Sequence Analysis
In the analysis of purified protein products, especially protein products, it is necessary to verify the ends of proteins to ensure the accuracy of the N-terminal and C-terminal sequences of the expressed and purified products. The Edman degradation method is one of the most mature methods for N-terminal sequence analysis of proteins and is widely used. BGI offers N-terminal sequencing services for purified protein products, antibodies, and protein vaccines using the Shimadzu Edman sequencing system. Our sequencing system can determine the sequence information of the first 30 amino acids at the N-terminus. With a specific protein loading system, it can determine 60-70 amino acids at the N-terminus. BGI has also established an N-terminal sequencing platform using advanced LC-MS/MS technology, capable of sequencing blocked and modified protein termini, complementing the Edman degradation method to ensure the smooth progress of N-terminal sequencing services.
Prior to protein sequencing, the protein samples are first separated by SDS-PAGE to ensure that the purity of the samples meets the sequencing requirements; then the protein samples on SDS-PAGE are transferred to a PVDF membrane; staining is performed to identify and cut out the protein bands; the Edman sequencer is used to analyze the protein samples obtained from the PVDF membrane. Edman degradation sequencing involves a cyclic reaction starting from the N-terminal of the protein to identify each amino acid type, thereby determining the N-terminal sequence of the protein. Each Edman sequencing reaction includes three steps: the first step involves, under alkaline conditions, the binding of PITC to the free amino group at the protein's N-terminus; the second step is the cleavage of the N-terminal residue in an acidic solution; the third step involves converting the PITC-bound residue into a more stable PTH residue, which is analyzed by online HPLC, determining the amino acid type based on the elution time.
Edman sequencing reaction process
Protein N-terminal sequencing service process based on Edman degradation
Edman sequencing experiment flowchart
Application areas
• Verification of protein expression products:Whether the insertion site and expression order of recombinant proteins are correct;
• Verification of protein products during cell line establishment and fermentation:Verify whether the N-terminal methionine and signal peptide of protein products during cell line establishment and fermentation are correctly processed;
• Protein degradation/enzyme cleavage analysis:Analyze the N-terminal of new protein fragments formed by degradation/enzyme cleavage to determine the cleavage/enzyme cleavage sites;
• De-novo sequencing:Analyze novel protein sequences not present in protein databases.
About the sample
Preparation process for PVDF membrane samples 1. Sample electrophoresis and transfer
a. Perform 1D or 2D gel electrophoresis on protein samples
b. Transfer samples from the protein gel to the PVDF membrane; Note: Do not use nitrocellulose membranes
c. During this process, wear gloves and a hairnet to avoid contamination from keratin affecting sequencing results
2. Staining of the PVDF membrane
a. Use Coomassie Brilliant Blue or Ponceau S for staining the PVDF membrane; Note: Do not use silver staining
b. After staining, wash the PVDF membrane with double-distilled water
c. If glycine is present in the transfer buffer, wash the PVDF membrane multiple times to avoid interference with subsequent analysis
d. For more detailed sample preparation steps, see the Edman sequencing sample preparation protocol
3. Acquisition of target protein bands
a. After staining the PVDF membrane, the target band is clearly visible. Use a clean scalpel to cut out the target protein band
b. If protein quantity allows, prepare 2-3 target bands to increase the amount of target protein
4. Sample transportation
Seal the cut protein bands in a package and transport with ice packs
Preparation process for solution samples 1. Protein purity and quantity
a. Required sample amount: 1-10 μg
b. Sample purity must be >90%
c. Avoid using surfactants in the buffer and minimize the salt concentration in the solution
d. Tris, glycine, guanidine, glycerol, sucrose, ethanolamine, SDS, Triton X-100, Tween, and other detergents, ammonium sulfate, and other ammonium salts can affect subsequent amino acid identification and should be avoided during sample preparation
e. Wear a mask and hairnet throughout the sample preparation process to avoid keratin contamination
2. Protein sample transportation
a. Liquid samples are recommended to be transported with dry ice
b. Liquid samples can also be transported with ice packs after freeze-drying or vacuum-drying
Research case studies
As shown in the figure below, Edman sequencing provides detailed information about identified amino acids.
Edman sequencing research case
Project report in Chinese/English
In the technical report, BGI provides a detailed bilingual (Chinese and English) technical report, which includes:
1. Experimental steps (in Chinese and English)
2. Related mass spectrometry parameters (in Chinese and English)
3. Detailed information on protein N-terminal sequencing
4. Mass spectrometry images
5. Raw data
