Sequencing of monoclonal antibodies and CDRs

Proteome Factory has long experience in providing full services for complete antibody and CDR sequencing and characterization using a combination of N-terminal and internal Edman protein sequencing, and high resolution MS and MS/MS mass spectrometry followed by de novo peptide sequencing.

Some of our equipment used for de novo sequencing of mABs and other proteins are

• nanoHPLC-ESI-MS/MS instrumentation (Orbitrap and FTICR)
• Edman sequencers
• micropreparative HPLC with fraction collector and parallel online high resolution MS and MS/MS detection 
• bioinformatic pipeline

LC and HC fragments of purified antibody are separately digested by several cleavage methods into peptides to get overlapping amino acid peptide sequences of the protein. The peptide mixtures are analyzed directly by nanoHPLC-ESI-MS/MS using only high resolution mass spectrometers to generate high mass accuracy/high confidence data.

Further preparative HPLC separation and fractionation of peptides is done with by HPLC with parallel ESI-MS/MS monitoring to trace peptides of interest for internal Edman sequencing and further MS/MS characterisazion.

MS/MS peptide fragmentation data is evaluated by de novo peptide sequencing and/or protein database search using Proteome Factory's proprietary Full Antibody Sequencing Software and Database. The full protein amino acid sequence is derived by putting together the peptide puzzle of overlapping peptide sequences.

Optionally validation of protein sequence is performed by SRM (selected reaction monitoring) analyses of peptide sequences which have to be validated by another analytical method.