Antibody Sequencing

De Novo Antibody Protein Sequencing

De novo protein sequencing of monoclonal antibodies is required when the cDNA or the original cell line is not available, or when characterization of posttranslational modifications is needed to verify antibody integrity and effectiveness.

Omics Technologies is proud to offer a series of unrivalled de novo antibody sequencing services for research, diagnostic and therapeutic industries. Omics Technologies has developed the proprietary pipeline for antibody protein de novo seqeuncing, MyProt™-ABSeqs™, which is composed by conventional shotgun based database-dependent mass spectrometry sequencing method, and database-independent next generation proteomics de novo sequencing technologies, together with variant identification and de novo PTM profiling capacities.

1) Database-dependent Sequencing of the conserved regions:

The V and J and C gene segments for different well studied host species are available in public databases, howver, there will be a good amount of mutations or variants in the sequences coming up during the maturation of the antibody. Our database search software platform are designed to tolerate such variant with non-compromised capability to sequence those regions and map these mutated peptides to the germline with FDR < 0.1%, meanwhile identify these mutations.

The success of an antibody sequencing project is based on our complete coverage MyProt™-CompleteID proteome profiling services. Different proteases (typically 5 or 6, depends on your antibody) will be applied and typically over 100 different MS/MS spectra are generated to cover each amino acid (AA) position, which is equivalent to 100X coverage. If we do not reach 100% coverage with at least 2 different peptides for each AA position, you will not be charged.

2) Database-independent De Novo sequencing of the highly variable region:

D segments of the antibody are heavily modified during anbitody maturation with only a small portion of the sqeuence remain in the matured antibody. The seqeunce of the D segment has to be sequence through a de novo approach.

One of the biggest advantage of MyProt™-CompleteID proteome profiling service is to generate a massive amount of peptides covering each AA position. Through commercial softwares and our proprietary bioinformatic and biostatistic packages and scripts, Omics Technologies can assign such massive amont of peptides to repeated cover each AA precisly with the FDR < 0.5%. Our de novo sequencing pipeline is so powerful that we could routinely read out the entire sequence of any unknown protein or protein fragments within one run at an unbeatablely small FDR.

1) Sample preparation – antibody preparation; protein quantification.

2) Sample digestion – Reduction and alkylation followed by in-solution digestion with 5 or 6 different proteases (in parallel groups) and C18 cleaning.

3) 2D fractionation – bRPLC separation of the C18 cleaned samples into 96 fractions, and further pooled into 24 fractions according to MyProt™-iPooling algorithm (based on peptide hydrophobicity, IEP, etc.)

4) Nano LC/MS/MS – 50 nL/min liquid chromatography to enable super high ionization efficiency for downstream Mass Spectrometry Analysis using the state-of-the-art Orbitrap Mass Spectrometer. 24 fractions will be analyzed in 24 Mass Spectrometry runs.

5) Protein profiling – Protein database searching with Mascot, Sequest HT, PEAKS de novo approaches, etc.

6) Data analysis – Data validation, visualization and quantification using commercial softwares and Omics Technologies's unique R packages and scripts.

7) Report generation – Report will be sent to you in Excel format as well as a summary in PDF format. We will also provide you any details you need for your papers' MATERIALS AND METHODS section. We will make sure you understand your result and help you with your paper writing with free follow-up services.

1, purified antibody

2, Customized sample types (please contact us to discuss)

Please refer to our Omics Technologies Proteomics Sample Preparation and Shipping Guidelines for instructions on how to prepare your samples with less contamination for Mass Spectrometry Analysis.