The primary structure of a protein is the linear sequence of amino acids that make up the polypeptide chain of the protein. Protein sequencing, protein sequence analysis, primary structure analysis, and amino acid sequence analysis are processes to determine the complete amino acid sequence of a protein. The information obtained through protein sequencing has numerous valuable applications, including: 1. Identification of proteins; 2. Designing probes for molecular cloning; 3. Synthesizing peptides that can be used as immunogens, etc.
There are two main methods commonly used for protein sequencing services: Edman degradation (Harvey and Ferrier, 2011; Bauer et al., 1997) and mass spectrometry (MS) (Sahukar et al., 2016). Currently, the most widely used methods for protein sequencing and identification are mass spectrometry, while Edman degradation is one of the most important methods for characterizing the N-terminus of proteins. Biotai Pike Biotechnology usesnano LC-MS/MS nano liquid chromatography combined with tandem mass spectrometryandShimadzu's Edman degradation sequencing systemto analyze protein sequences, providing mass spectrometry-based protein sequencing analysis services, including proteinamino acid composition analysis,N-terminal sequencing,C-terminal sequencingandfull sequence analysis, as well asEdman degradation-based protein N-terminal sequence analysis services. For proteins with unknown theoretical sequences, we provide de novo sequencing-basedprotein de novo sequencing services, to analyze protein sequences.
Mass spectrometry for protein sequence analysis, compared to Edman degradation, has advantages such as greater sensitivity, faster peptide cleavage, and the ability to identify proteins with terminal modifications or closures. Biotai Pike Biotechnology utilizes the current high-resolution mass spectrometry technology platform to provide mass spectrometry-based protein sequence analysis services, achieving 100% coverage of the target protein sequence. This analysis can be used to confirm whether the recombinant protein is fully expressed and to detect any cleavage during the recombinant protein expression process.
In the process of protein sample sequence analysis, Trypsin is generally used for enzymatic digestion of proteins, with an identified peptide coverage rate of about 60%. To obtain the full sequence information of the target protein, Biotai Pike Biotechnology selects six commonly used proteases (Trypsin, Chymotrypsin, Asp-N, Glu-C, Lys-C, and Lys-N) to perform enzymatic digestion and mass spectrometry of the target protein. Through the sequencing of fragmented peptides, the complete protein sequence is determined 100%. Biotai Pike uses Thermo's newly launched Obitrap Fusion Lumos mass spectrometer for protein sample sequence analysis. The Obitrap Fusion Lumos mass spectrometer is currently the highest resolution and sensitivity mass spectrometer, ensuring the sensitivity of low-abundance peptide fragment identification. During peptide fragmentation, the combined HCD and ETD modes ensure the integrity of peptide fragments. This allows for N-terminal, C-terminal, and full-length sequence analysis of protein samples.
Protein sequencing diagram
Edman degradation method for protein N-terminal sequence analysis
Edman degradation sequencing method is a chemical method that can obtain N-terminal amino acid sequence information of proteins, developed by Pehr Edman in the early 1950s. This method allows labeling and analysis of the N-terminal amino acid sequence without disturbing the peptide bonds (Hunkapiller, 1988; Liu et al., 2016).
Edman degradation method is widely used in protein sample N-terminal sequence analysis. However, in practical applications, the Edman degradation method has certain limitations. For example, it cannot be used for sequence analysis of proteins with N-terminal closures or chemical modifications. In such cases, advanced nano LC-MS/MS nano liquid chromatography combined with tandem mass spectrometry platforms can be used for N-terminal sequence mass spectrometry, which can analyze sequences of proteins with terminal closures and modifications, complementing the Edman degradation method to ensure smooth N-terminal sequencing services. Biotai Pike Biotechnology uses Shimadzu's Edman sequencing system to provide N-terminal sequencing services for purified protein products, protein vaccines, and various protein samples for researchers and scientific customers. Our sequencing system can determine sequence information for up to 30 amino acids at the N-terminus.
Sample preparation
There are many methods available for sample preparation, and no particular method is the best for sample preparation. Each method has its own advantages and disadvantages. The best method for preparing a specific sample can be determined by combining laboratory experience and common methods, and by considering your own requirements for yield, purity, cost, and speed. The most common methods for preparing protein sequencing samples include:
1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
2. High-performance liquid chromatography (HPLC)
3. Capillary electrophoresis (CE)
*Factors to consider when selecting a sample preparation method include: 1) the required experimental cycle; 2) the purity requirements of the sample; 3) the quality of the protein needed. If you do not currently have the conditions for sample preparation, Biotai Pike Biotechnology offers one-stop protein analysis services that include sample preparation services. Feel free to contact us for consultation.
About samples
Biotai Pike Biotechnology's one-stop protein sequencing services can analyze protein sequences from samples in various states such as PVDF membranes, gel spots, gel strips, and solutions. Sample transportation is a common concern; cut protein bands can be sealed and packed, and transported using ice packs. Solution samples can be vacuum-dried or freeze-dried, then transported using ice packs or dry ice. Feel free to contact us for consultation.
