In Silico Proteome Cleavage Reveals Iterative Digestion Strategy for High Sequence Coverage
Theoretical upper limits of coverage upon digestion with various cleavage agents using the iMED-FASP strategy. Iterative cleavage of the proteome starting with the rarest amino acids first results in the greatest theoretical proteome coverage of 92.9%. The reversed sequence of cleavage provides a minimal improvement to theoretical proteome coverage. Peptides were filtered after each digest keeping those with MW > 5 kDa for additional digestion. The final “flowthrough” peptides were filtered keeping only sequences with at least 7 residues.
Theoretical coverage limit (%)
CNBr, LysC, trypsin
NTCB, CNBr, LysC, trypsin
TrpC, NTCB, CNBr, ArgC, GluC, trypsin
TrpC, NTCB, CNBr, ArgC, AspN, GluC, trypsin
Trypsin, GluC, AspN, ArgC, CNBr, NTCB, TrpCa
Reversed order of cleavage starting with the most common residues instead of the rarest residues.