LC-MS/MS Protein Full Sequence Verification
With the advancement of modern pharmaceutical industry, sequence analysis has become an important part of the development and quality control of biological drugs such as proteins, peptides, antibodies, vaccines, and collagen-based medical devices. Full sequence verification analysis of proteins plays a key role in ensuring the complete and accurate expression of recombinant protein-peptide biological products, thereby ensuring product quality and efficacy.
BTP BioScience has established a protein sequencing platform based on the dual quality certification system of CNAS/ISO9001. This platform is equipped with multiple N-terminal sequence analyzers, ultra-high-performance liquid chromatography high-resolution mass spectrometry, and a de novo protein sequencing algorithm platform optimized through multiple iterations. Combining advanced analytical equipment and algorithms, BTP can perform full sequence verification analysis on proteins to ensure their integrity and accuracy, meeting your research and quality control needs.
LC-MS/MS Protein Full Sequence Verification Analysis:
The general process of protein full sequence verification analysis involves enzymatic digestion of protein molecules using specific proteases, resulting in a complex mixture of peptide fragments. This mixture is then subjected to mass spectrometry analysis through an LC-MS/MS platform.
Firstly, the sample undergoes pretreatment, where at least five proteases are selected for digestion based on the theoretical full-length amino acid sequence and mass spectrometry-grade protease cleavage site information to improve digestion efficiency and achieve higher coverage. In actual experiments, suitable proteases or combinations, such as Trypsin, Chymotrypsin, Asp-N, Glu-C, Lys-C, and Lys-N, can be selected for target protein digestion. Next, the digested peptide mixture is analyzed using the Nano-LC-MS/MS platform to obtain total ion chromatograms and raw mass spectral data. Then, a local database is established with the theoretical sequence of the protein, and each identified peptide from the digestions is matched and calculated using specialized software. The protein sequence is assembled using the complementarity between cleavage sites, ultimately achieving full sequence verification.
Case Illustration:
Antibody Light Chain Full Sequence Verification Analysis Illustration
General Process:
1. Use a combination of multiple proteases for digestion to improve digestion efficiency and obtain sequence information for each amino acid of the protein.
2. Analyze the sample using the Nano-LC-MS/MS high-resolution platform.
3. Compare the obtained mass-to-charge ratio information with the database established from the theoretical sequence.
4. Assemble the peptides identified from each digestion method to obtain information on whether the protein is completely and correctly expressed.
Experimental Instruments:
- Liquid Chromatography System (Easy-nLC1200)
- Electrospray-Combination Quadrupole-Orbitrap Mass Spectrometer (Q Exactive™ Hybrid Quadrupole-Orbitrap™ Mass Spectrometer)
Applications:
- Amino Acid Full Sequence Verification Analysis of Biological Products like Proteins, Peptides, Antibodies, Vaccines, and Collagen
- Protein Post-Translational and Chemical Modification Analysis
- Amino Acid Sequence Mutation Analysis of Biological Products like Proteins, Peptides, Antibodies, Vaccines, and Collagen
Frequently Asked Questions:
Question 1: What does coverage reflect? What should be done if coverage is less than 100%?
Answer:Coverage refers to the ratio of the sequenced part of the sequence to the total sequence size. It reflects the coverage of our sequencing results on the protein. A coverage of 100% indicates that the sequence obtained after enzymatic digestion matches the target sequence perfectly. If the coverage is less than 100%, we will review the protein data and sequence to check how much sequence is missing and which theoretical peptides were not detected. Based on the actual results, we will decide whether to continue using the existing method and enzymatic treatment to retrieve the missing peptides, or if the protein sample itself has mutated, requiring de novo sequencing analysis.
Question 2: How to prepare protein samples if the protein to be sequenced has a relatively small molecular weight?
Answer:If the protein has a relatively small molecular weight, a higher-concentration SDS-PAGE gel can be used to separate the protein, followed by Coomassie Brilliant Blue staining of the protein gel. Then, the protein bands corresponding to the target protein size can be cut out. These cut-out gel strips are used for mass spectrometry identification.
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