Pegylation Of A Protein

The use of proteins and peptides as therapeutic agents is widespread, but their effectiveness is often limited by their stability and how long they remain in the body. To overcome these limitations, PEG-protein conjugates have been developed. The addition of a polyethylene glycol (PEG) molecule to a protein enhances its therapeutic potential by reducing its removal by the kidneys, increasing its resistance to enzymatic degradation, and lowering the likelihood of an immune response.

For consistent and effective biotherapeutics, it's crucial that PEGylation—the attachment of PEG—occurs at a specific, predetermined site on the protein. Pinpointing this exact location is vital for thoroughly characterizing the drug. Traditionally, peptide mapping has been employed to identify any modified peptides. However, directly confirming the specific amino acid to which PEG is attached within a peptide has proven difficult.

We've developed a novel high-resolution mass spectrometry (HRMS) method to sequence PEGylated tryptic peptides, specifically those where a cysteine residue is linked to PEG-maleimide. This method leverages in-source collision-induced dissociation (SID) and higher-energy collisional dissociation (HCD) MS2 analysis of the PEG conjugates.