How to Achieve Protein Sequencing?

Protein sequencing mainly refers to the determination of the primary structure of proteins. The primary structure of a protein is the sequence of amino acids that make up the protein. Protein amino acid sequence analysis is the process of determining the full amino acid sequence of a protein. The determination of protein amino acid sequence is the basis of protein chemistry research. Since the determination of the primary structure of insulin by F. Sanger in 1953, the primary structures of about 100,000 different proteins are now known.

The information obtained from protein sequencing has many uses, including protein identification, synthesis of peptides that can be used as immunogens, development of generic antibody products for therapeutic use, and antibody drug development based on commercially available antibody reagents. Current protein sequencing methods are divided into three main categories: PCR amplification-based protein sequencing, Edman degradation sequencing, and mass spectrometry-based protein sequencing.

PCR amplification-based protein sequencing is the use of DNA or RNA expressed in cells for gene sequencing, which is then converted into amino acid sequences of proteins according to the amino acid codon table. It is essentially a gene sequencing technique.

Edman degradation sequencing is an early developed protein sequencing technique that uses chemical methods to sequentially degrade amino acids from the N-terminal end of proteins and then uses high-performance liquid chromatography to identify the amino acids. However, this method can only be used to identify the N-terminal amino acid residues of proteins and peptides (usually a few to a dozen residues, up to a maximum of forty residues) and cannot be used for full sequence determination of large proteins. In addition, the Edman degradation method has some limitations. For example, protein sequences of N-terminal closure or chemical modifications cannot be analyzed using the Edman degradation method.

The most widely used method for protein sequencing is mass spectrometry. Compared to Edman degradation methods, it is more sensitive, can cleave peptides faster, and can identify proteins with closed or modified ends. Mass spectrometry-based protein sequencing strategies can be divided into two main categories: top-down strategies and bottom-up strategies. The top-down strategy does not require degradation of the protein, and the intact protein is directly analyzed using LC-MS to determine its sequence based on fragment ions in the spectrum. The bottom-up strategy is to hydrolyze the protein into peptides, detect the peptides by LC-MS, and then sequence the peptides from scratch as well as sequence splicing to obtain the complete protein sequence.

Creative Proteomics has established a protein sequencing platform to effectively address the problems of low coverage and accuracy of protein sequencing. Multiple non-specific proteases are used to sequentially digest proteins to increase the protein digestion peptide species and overlap, thus improving the coverage of protein sequencing. In addition, a sequence splicing algorithm was developed to assemble and splice protein sequences based on the score value and number of occurrences of each amino acid in the peptide sequences obtained from de novo sequencing, which significantly improved the accuracy of protein sequence determination.

Creative Proteomics has gradually grown into an integrated service provider with targeted lipidomics and untargeted lipidomics analysis services for researchers in pharmaceutical, biotechnology, agriculture, and nutrition industries.