Post Translational Modification Analysis Service.
Identify the position and relative abundance of post-translational modifications (PTMs) using LC-MS. This service is optimized for monoclonal antibodies (mAbs) and related forms.
How PTM Analysis by LC-MS Works.
The most widely accepted method for analysis of antibody post-translational modifications uses high pressure liquid chromatography, coupled with mass spectrometry. The primary structure is best evaluated by first digesting the protein into peptides prior to analysis – ‘bottom up’ mass spectrometry. The masses of peptides and fragments of peptides can be matched to theoretical masses from the expected sequence in a process called peptide mapping. Since some amino acid residues are modified after translation, their masses will be affected predictably – for instance deamidation of Asn and Gln, oxidation of Met and Trp, succinimide (N), pyroglutamate. Accounting for these mass shifts allows us to map the modified peptide and its fragments to the expected sequence, and thereby identify the type and location of PTM.
Since both the modified and unmodified forms of the peptide will be detected by mass spectrometry, it is also possible to compare the quantity of modified vs unmodified peptides by looking at their peak areas on the MS1 spectrum.
Why PTM Analysis Matters.
Certain antibody sequences are more prone to post-translational modifications (PTM) than others. Antibody discovery scientists and antibody engineers may select lead antibodies that are less prone to PTM in order to avoid downstream challenges – early and fast characterization by LC-MS helps this process.
Antibody production scientists must determine which production, handling and storage conditions are more likely to cause PTM. In this case, we help our clients to compare between samples produced and handled under various conditions to see which ones have relatively more or less of these PTM.
Which PTMs to look for.
Aside from Glycosylation, the most common PTM that affect antibodies are:
- N terminal cyclization
- C terminal lysine truncation
- Deamidation (Asn&Gln)
- Oxidation(Met & Trp)
- Succinimide (N)
- Pyroglutimate
Unless otherwise specified, these are included in our analysis. You may select from a list of common PTM, or specify a custom PTM (the amino acids affected and the mass shift must be specified). Glycosylation analysis is a separate service, which can be conducted in parallel.
Focused PTM analysis.
You may choose to scan the whole antibody sequence for all modifications, and quantify each of them. You may choose to analyze the whole antibody for one specific type of modification and quantify its presence at various locations. Or you may choose to focus your analysis on one specific location within the protein. More focused analyses can be completed more efficiently.
Getting Started.
Requirements
- Amino acid sequence(s) in FASTA format
- 20 μg per analysis per sample
- >90% purity
- Monoclonal antibody (any isotype, format or fragment)
- Optional: List of PTM to include in analysis
- Optional: specific location to analyze
Add Ons:
- D-isomerization analysis
Deliverables
- Sequence annotated with PTM names
- Prevalence of PTM at each AA position (calculated by peak area)
- Identification of c-terminal or n-terminal truncation
- Optional: comparison of samples
- Add-on: D-isomerization analysis
Timeline
Screening
Talk to Our Scientists.
We Have Sequenced 9000+ Antibodies and We Are Eager to Help You.
Through next generation protein sequencing, Rapid Novor enables reliable discovery and development of novel reagents, diagnostics, and therapeutics. Thanks to our Next Generation Protein Sequencing and antibody discovery services, researchers have furthered thousands of projects, patented antibody therapeutics, and developed the first recombinant polyclonal antibody diagnostics.
Talk to Our Scientists.
We Have Sequenced 9000+ Antibodies and We Are Eager to Help You.
Through next generation protein sequencing, Rapid Novor enables timely and reliable discovery and development of novel reagents, diagnostics, and therapeutics. Thanks to our Next Generation Protein Sequencing and antibody discovery services, researchers have furthered thousands of projects, patented antibody therapeutics, and ran the first recombinant polyclonal antibody diagnostics
Talk to our scientists. We have sequenced over 9000+ antibodies and we are eager to help you.
Other Antibody Characterization Services.
PTM Analysis
- Sequence annotated with PTM names
- Prevalence of PTM at each AA position (calculated by peak area)
- Identification of c-terminal or n-terminal truncation
- [comparison of samples]
- [Optional D-isomerization analysis]
Sequence Variant Assessment
- List of peptides that closely match the expected sequence
- % of each variant (by peak area)
- List of peptides that match common contaminants
- Rough assessment of quantity of non-matching peptides
Glycosylation Analysis
- Glycan site occupancy report
- Glycan profile (i.e. g0, g0f, g1, g1f, etc. – % values that sum to 100)
Peptide Mapping
- Peptide map
- % coverage achieved
- List of observed peptides
- Interactive coverage viewer and coverage map
- Any other observations for further investigation
Intact Mass, Custom Analyses
- Please inquire about intact mass analysis, subunit analysis, etc.
Other Services.
Antibody Sequencing & Discovery Services.
REmAb®
Monoclonal antibody sequencing from small antibodysamples, no need for hybridoma or DNA information. Full sequence in record time.
Explore REmAb
REpAb®
Polyclonal antibody sequencing and antibody discovery service. Sequence antibodies from blood or a polyclonal mixture.
Explore REpAb
MATCHmAb™
Antibody verification by real-time peptide mapping. Antibody sequence confirmation service for reproducibility.
Explore MATCHmAb
HDX-MS
Mass spec based epitope mapping. Epitope mapping for identify the binding site of an antibody to its corresponding antigen with the highest confidence and resolution.
Explore Epitope Mapping
SPR Analysis
Label-free evaluation of antibody-antigen binding relies on a Surface Plasmon Resonance technology. Combined with our expertise in antibody interactions, this results in high sensitivity and the lowest noise even in complex analytes.
Explore SPR Analysis
NovorIg™
Quantify the immune response by accurately profiling the vast antibody-protein repertoire and its different isotypes, and subtypes in serum over time.
Explore Immune Response Profiling