Alkyations and digestion of proteins
Digestion of proteins
The Biomolecular Research Facility performs enzymatic and cyanogen bromide digestions of proteins, separates the resultant peptides and sequence them by mass spectrometry or Edman sequencing.
The most common means of digestion are with lysyl peptidase, which cuts after lysine residues; This is the preferred enzyme for producing peptide for Edman sequencing. Trypsin, which cuts after lysine and arginine residues is the standard enzyme used in mass spectrometry analysis and tryptic digestion is part of the standard protein analysis by mass spectrometry. Cyanogen bromide which cuts after methionine. is another favoured digestion.
Prior to digestion or sequencing, it is preferable to reduce and alkylate the protein to eliminate disulfide bridges, and hence cross linked peptides and protect cysteine; without alkylation, cysteine cannot be identified during Edman sequencing. The preferred alkylating reagent for Edman sequencing is N-isopropyliodoacetamide: iodoacetate and iodoacetamide give derivatives which are not easily distinguished from other amino acids during Edman sequencing. We can alkylate proteins after transfer to PVDF.