Peptide cleavage

Why cleave peptides to proteins?

Peptides must be generated prior to MS identification since peptides fragment well in CID. For proteomics, peptides are typically generated from liquid solutions, gel pieces, and membranes.

Peptide Generation

Peptides can be efficiently generated by enzymatic or chemical cleavage methods compatible with MS. The most common technique is proteolytic cleavage followed by chemical cleavage.

Proteolytic Cleavage

In proteolytic cleavage, proteinases (proteases or peptidases) are enzymes which break peptide bonds of proteins.

Trypsin

Trypsin is the most widely used endoproteinase for protein identification by PMF for the following reasons:
• Cleaves on C-terminal side of basic amino acids K and R when they are not followed by P.
• Wide distribution of peptide masses (500 - 4000 Da) useful for MS analysis.
• Creates 2 charge sites which are very useful for ESI
• Positioning of charges on N and C terminals simplifies interpretation of CID spectra

Chymotrypsin

Mainly cleaves on C-terminal sites of T, Y, F, and L residues. It is difficult to predict cleavage fragments and therefore it is not very useful for PMF. It is poorly specific due to large number of possible cleavage sites. However, due to high sequence coverage, it is considered very useful for characterizing PTMs.

Pepsin

Pepsin is a non-specific acidic protease. It can cleave both N and C terminals of aromatic amino acids and hydrophobic residues. Not useful for PMF but very useful for characterization of PTMs. It can also be used to digest hydrophobic membrane proteins that cannot be attacked by trypsin due to lack of basic residues.

Chemical Cleavage

Chemical cleavage of proteins is very important for Edman sequencing. Although chemical cleavage is very specific, it requires cleanup before MS.

Cyanogen Bromide

• It cleaves very specifically at methionyl residues in the peptide backbone.
• This method has been successfully used with membrane proteins.
• Since M is often situated in hydrophobic regions of proteins, digestion using CNBr can generate peptides with decreased hydrophobicity, ultimately enabling protein analysis.

Acidic Hydrolysis

External factors can have enormous influence on the cleavage.