About glycosylation

Glycosylation refers to the attachment of sugar moieties (glycans) at sites on the protein sequence to produce glycoproteins.

Monoclonal antibodies (mAbs) are an important example of glycoproteins, which contain a conserved N-linked glycosylation site on the Fc region in the CH2 domain. This modification has many known physical functions in the molecule and characterization is extremely important.

There are two main types of glycosylations:

  1. N-linked glycosylation occurs as a linkage between the amino sugar N-acetylglucosamine (GlcNAc) and asparagine (N) at the consensus motif N−X−S/T, where S is serine, T is threonine, and X is any amino acid except proline.
  2. O-linked glycosylation usually occurs as a linkage between the amino sugar N-acetylgalactosamine (GalNAc) and serine (S) or threonine (T).

The BioPharma Finder application recognizes over 200 different glycan structures so that you can potentially identify unexpected modifications in your protein sequence.

The "Glycans" appendix lists all of the N-linked glycans included in the default modifications list, N-linked glycans included in the glycan-specific search (sorted by host cell-line type), and O-linked glycans included in the glycan-specific search (peptide mapping analysis only).

NOTE

The algorithm that detects possible glycosylation sites was designed for use on intact proteins. If it is applied to digested peptides, it might fail to identify motifs that have been truncated by a cleavage.

To address this issue when using a peptide as a target sequence, append an amino acid to the sequence to complete the motif, and then define and apply a custom modification that subtracts the mass of that amino acid.

Assign a glycosylation to your protein sequence

Procedure

  1. In the Protein Sequence Editor, select the title bar for Variable Modifications for Intact and Peptide Analysis.
  2. Variable Modifications for Intact and Peptide Analysis pane
    Variable Modifications for Intact and Peptide Analysis pane

  3. NOTE

    To edit the parameters in this pane, you must set the category for the protein sequence to Peptide Mapping, Intact Protein, or Unknown.

  4. In the N, O Glycan drop-down list in the Glycosylation area, select the type of glycosylation (or none) to apply to the N-linked glycans and O-linked glycans:
  5. None: Does not add any search for glycosylations.
  6. CHO: Adds a search for glycosylations to Chinese hamster ovary (CHO) glycans.
  7. Human: Adds a search for glycosylations to human glycans.

  8. NOTE

    The application supports O-linked glycans for peptide mapping analysis only.

  9. Select Save or Save as New in the command bar to save the modifications. Or, if you do not want to save the modifications to the sequence, select Cancel.