The workflow is tested with the Waters’ IgG-I antibody standard (product number 186006552). The full sequences for both the heavy and light chains are fully recovered (Figure 1) with high coverage for each amino acid.
Figure 1. Software’s interactive coverage view for light chain. Each AA is covered by multiple peptides. Colors indicate peptides from different enzyme digests. Paled color indicates the peptide’s peak area < 0.1%. The ruler at the top highlights the CDR, variable, and constant regions of the antibody. Heavy chain coverage is similar.
Compared to the sequence provided by Waters, two variations were discovered on the heavy chain. The first replaces two amino acids at 49-50 from MG to GM, and the second replaces three amino acids at 68-70 from SIT to TIS. Both changes are on the variable region and are supported by strong MS/MS signal peaks (Figures 2). Since the variations do not change the intact mass of any tryptic peptide, and only slightly change the MS/MS spectra, they can only be picked up with de novo peptide sequencing. Peptide mapping or a homology-based sequencing would have failed in detecting these mutations.
Figure 2. Evidential spectra for the mutations at heavy chain 49-50 (published MG vs our sequenced GM), and 68-70 (published SIT vs. our sequenced TIS). These mutations could not have been detected with a homology-based method as the published wrong sequences already match the spectra significantly.
Additionally, six glycosylation forms were identified (Figure 3). Five out of the six identified forms have slightly different retention time. Thus, they are not the result of in source fragmentation during MS.
Figure 3. Glycosylation forms identified by the workflow.