PEAKS AB 3.0 Software New Features

A new feature introduced in PEAKS AB 3.0 is the Glycan Profiling tool. This tool performs in-depth glycan profiling for N-linked sites identified in the heavy and/or light chains of the antibody. In addition to accurate glycopeptides mapping to the assembled antibody sequence, enzyme-based glycan profiling displays the composition and relative abundance of each glycan at a selected glycosylation site. Glycan composition and structure annotation are provided within each glycopeptide spectrum and are based on glycan fragment ions that match to an N-linked glycan database. Accurate localisation of the glycan at each N-linked site is achieved by identifying fragment ions of glycan moieties associated with the peptide backbone.

In PEAKS AB 3.0 protein samples other than antibody can be sequenced. The box next to “Use reference sequence (provide one or two FASTA sequence)” should be checked and the target sequence can be added to the white box below.

Sequence validation and manual editing is an essential step of any protein sequencing project. Sequencing errors can arise from background contamination of homologous proteins or from low coverage and data quality. By selecting 3 or more amino acids, the user can now select “Find homology sequences” to reveal any alternative peptide sequences that could map to that region.

The alternative peptide sequences displayed are colour coded based on local confidence scores for each amino acid, for quick assessment of peptide sequence quality. By selecting the amino acids to be edited, then changing to the corrected sequence, clicking “Confirm changes” will edit the reference sequence. Applying these changes from the Peptide Mapping window will generate a new sequencing result node after a second round of sequence assembly. 

The PEAKS AB 3.0 advanced deconvolution algorithm enables automated, efficient, and accurate intact mass analysis. Intact mass measurements of proteins are performed to validate the assembled sequence and presence of any modifications such as N-terminal pyro-glutamate (Gln->Pyro-Glu), C-terminal lysine truncation (1*Lys-Trunc), and N-linked glycosylation (1*A2G0F), as shown below.