单抗从头测序(Complete De Novo Sequencing of mAb & Other Proteins)

ProtTech®提供单抗全序列从头测序服务，我们建立了抗体从头测序的一系列方法并开发了十多个专有软件，已有完善的数据分析平台，我们能保证测序的准确。

1：单抗轻重链肽段覆盖率达到。

2：首次检测氨基酸准确率达到99.5%以上，活性如果有差异，我们免费复测，找出可能的差异点，保证客户在CHO细胞表达的抗体活性与原来相同。

3：适用于 人源、小鼠源、大鼠源、兔源等的IgG和IgM。

4：样品要求：蛋白纯度95%以上，蛋白量500ug 以上，周期4周。

ProtTech offer the service for the complete de novo sequencing of mAb and other proteins. The service is based on the proprietary protein sequencing technology that we developed over many years. We guarantee a result with a sequencing coverage of the entire mAb molecules, a better than 99.5% accuracy and distinct assignment of Leu/Ile residues.

We also offer an optional confirmation service to confirm the correctness of the variable regions of the sequenced mAb. In this validation process, we express HV and LV region O in E. coli, and use LC/MS/MS peptide mapping to compare each variable region peptide from original and expressed proteins.  We gurantee a correctness of the mAb sequence after this confirmation process. Since we use E. coli as the expression host, the confirmation process is often much faster and lower cost than mammalian host expression. Since the sequence confirmation is based on MS and MS/MS of each peptide instead of ELISA assay, the sequence fidelity is much higher.

Procedure:

The complete mAb de novo sequencing is often a very complex process, involving more than ten different proteolytic digestions, several steps of chemical derivatizations, NanoLC-ESI-MS/MS data acquisition, bioinformatics analysis with more than a dozen  proprietary softwares, the manual spectra assignment, etc. In general the process contains the following steps: 1). Proteolytic digestion and chemical derivatization. 2). LCMS data acquisition. 3). Database search and peptide de novo sequencing. 4). Draft sequencing assembling. 5). Sequence gap filling. 6. Sequence error screening. 7). I/L determination. 8). Sequence validation.

Our mAb de novo sequencing technology can also apply to other proteins. However, changes in the process is often needed based on the nature of the protein.

Service Terms:

Sample requirement: Since more than ten different proteolytic reactions are carried out in the sequencing process, we prefer to have > 1mg mAb protein for each project, although ~500ug mAb sample is acceptable. A better than 90% purity is required. The main concern about the quality of a sample is immunoglobin contamination since there is no easy way to separate contaminating antibodies from the mAb of interest, and there is not easy to determine if a peptide from contaminating peptides or from the mAb of interest. The major source of such a contamination is the serum in cell culture media.

Turn-Around Time:

It often takes five week to sequence one mAb sample.