The protein sample identification process generally only uses Trypsin for enzymatic digestion of proteins, with an identified peptide coverage of about 60%. To obtain the complete sequence information of the target protein, BiotechPack Packer selects six commonly used proteases in protein identification (Trypsin, Chymotrypsin, Asp-N, Glu-C, Lys-C, and Lys-N) to perform enzymatic digestion and identification of the target protein. This allows for the determination of 100% of the protein sequence by splicing between peptide fragments, while obtaining more fragmented peptide segments. BiotechPack Packer applies the existing mass spectrometry technology platform to provide peptide mapping analysis based on LC-MS/MS. This analysis can be used to confirm whether the recombinant protein is fully expressed and to detect whether there is any breakage during the recombinant protein expression process. It is mainly used for confirming the primary structure of proteins. By using various protease digestion reactions and liquid chromatography-mass spectrometry, combined with proprietary analysis software, it can analyze mass spectrometry data of any non-specific protease digestion products. Thus, achieving 100% coverage of the determined target protein sequence.
Process of mass spectrometry peptide coverage/full sequence determination:
• First, use six proteases (Trypsin, Chymotrypsin, Asp-N, Glu-C, Lys-C, and Lys-N) to perform enzymatic digestion and identification of the target protein
• Then use nano LC-MS/MS or UV LC-MS/MS to analyze the samples
• Compare the obtained peptide coverage/full sequence with the theoretical sequence you provided
• Obtain the comparison results
Advantages of mass spectrometry peptide coverage/full sequence determination:
(1) The combination of specific and non-specific proteases can achieve 100% coverage for the identification of any protein (peptide).
(2) Confirmation of each peptide segment includes: 1) Based on the mass of the sample; 2) Based on the MS/MS fingerprint spectrum of the sample, making it more accurate than using only primary mass spectrometry for determination.
Sample requirements:
Both in-gel samples and solution samples can be used for the above sequencing
The higher the sample purity, the better, recommended to be over 80% protein
Required amount of target protein sample: 10-20ug
Protein gel samples can also be used for peptide coverage determination, but require special pre-treatment of the sample.
Case study:
Protein sample 100% sequence verification service
In the actual experimental process, following the principle of maximizing efficiency and minimizing costs, we will first analyze the theoretical sequence provided by the customer. The analysis process predicts peptide coverage based on several different protease cleavage sites. If 100% coverage of the full sequence can be achieved with just three proteases, the actual experimental process will first use these three proteases for enzymatic digestion and subsequent sequence analysis experiments, effectively reducing the experimental cost. Below is part of the data from a previous project. In this project, the customer expressed an antibody drug according to the theoretical sequence, and the primary sequence needed verification to prove that the expressed antibody matched the theoretical sequence. Some experimental data are shown as follows:
In the final report, we will provide detailed data on peptide coverage as shown in the diagram below.
Chinese/English project report
In the technical report, BiotechPack Packer will provide you with a detailed bilingual (Chinese and English) technical report, including:
1. Experimental steps (Chinese and English)
2. Relevant mass spectrometry parameters (Chinese and English)
3. Mass spectrometry images
4. Raw data
5. Biopharmaceutical peptide coverage information
One-stop service for biopharmaceutical peptide coverage determination
You only need to place an order and send the sample
BiotechPack Packer completes the one-stop service: sample processing - on-machine analysis - data analysis - project report
