Antibody Sequencing by Mass Spectrometry

A: In the ideal case, 50ug of monoclonal antibody.  We’ve worked with less, but to ensure accuracy and completeness, 50ug is the safest amount.  If the sample contains impurities, e.g. other antibodies or BSA, we can purify these out before sequencing.

A: With modern mass spectrometers, it’s possible to distinguish isoleucine from leucine using EThCD fragment spectra.  We wrote in a blog post about methods for I/L calling.  Methods that rely solely on fragmentation spectra can struggle with residues near the terminii of peptides, or stretches of I/L together.  Combining multiple lines of evidence helps improve this prediction.

A: De novo antibody sequencing by mass spectrometry does not rely on a reference sequence or database and as a result is species and isotype agnostic.  Most formats (e.g scFv, Fab) are compatible with sequencing.  One special exception is bispecific antibodies in which two distinct heavy chains and two distinct light chains are present.  In most cases, all four chains can be sequenced successfully, however the pairing information may be ambiguous.

A: Antibody sequencing by mass spectrometry is distinct from DNA sequencing in three very important ways:

a) Antibodies are digested into thousands of small peptides that are then analyzed by the mass spectrometer.  In contrast, DNA technology produces reads that are long (300bp+) so that only a small number are needed to cover the entire antibody or at least the variable region.

b) Going from raw images on the sequencer to DNA sequences is considered a solved problem, and tightly coupled to the sequencer run.  For mass spectrometry, interpreting a mass spectrum to recover a peptide sequence is a challenging problem that is undergoing continual improvement both on the instrument side and the software side.

c) In DNA sequencing, PCR is use to target specific sequences or amplify low abundance transcripts.  For proteomics, there is no amplification method so we have to work with the quantity of protein present in the sample.

A: Not all PTMs are created equal in the world of mass spectrometry.  Small, labile modifications, like oxidation, deamidation, or phosphorylation, can be easily detected since they result in small shifts in the mass spectrum, and both the modified and non-modified versions of the antibody are typically observed.  For large PTMs, like glycans, identification can be more challenging.  As we described in a recent blog post, determining that a glycan is present and recovering the sequence of the protein is achievable.  Determining the glycan structures, however, is typically a challenge that is outside the scope of de novo antibody sequencing.

A: Yes, mass spectrometry can be used to sequence polyclonal antibodies but the process is more complex than for monoclonal antibodies.  We recently outlined different methods for polyclonal antibody sequencing by mass spectrometry.