Review: Antibody Protein Sequence Analysis Using Mass Spectrometry

During the process of research and development of antibody drug molecules, an essential step is determining the sequence of the antibody. Mass spectrometry technologies have advanced so far as to make them the first choice in sequence analysis. This article describes three technologies which are frequently chosen for this purpose, namely:

Intact mass analysis
Peptide mapping
Protein de novo sequencing
While intact mass analysis and peptide mapping need the amino acid sequence of the antibody to be known beforehand, the third technique permits sequencing to be done directly using the LC-MS/MS data.

Intact Mass Analysis
Intact mass analysis is a technique which measures the molecular weight of the intact antibody protein, either in native form or as a separate determination of the heavy and light chain once the molecule is reduced. Antibody fragments may also be measured, such as for Fab or VH+CH1.

The determination of the primary sequence of the protein antibody is confirmed by finding the intact mass in this way. However, this does not permit variations in the sequence - such as leucine/isoleucine mutations or amino acid swapping - to be verified. Another use of this technique is to help evaluate the relative ratios of the glycosylated forms which have been expressed, because these modified forms are often bestowed with immunogenicity or biological activity by the sugar residues.

The procedure of an intact mass analysis is typically as below:

An LC-MS is carried out on the purified antibody, either in intact or fragment form, to obtain a full MS spectral readout
The high-charged full MS spectrum is deconvoluted
The data is analyzed and interpreted
The process is depicted in the following image which shows the target protein B with a high-charge envelope and the associated deconvolution
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