Dear Mr Thakkar,

A protein of Mw 18kDa is a fairly small protein and you should have no problem in achieving higher sequence coverages with trypsin.

Double digestions are usually applied for high Mw proteins (~500kDa, such as ApoB-100) or chemically modified proteins (Arg and Lys residues) where in these cases other enzymes are used (Chymotrypsin, GluC,...).

I wonder if you're doing everything right in your protocol as several other (experimental) factors could be leading to that result. For instance:

1) Is the ammonium carbonate (NH4HCO3) solution for trypsin aliqquots freshly prepared?

2) Trypsin does not tolerate detergents (SDS, triton X, ...)

3) Are you adding enough DTT? If your protein has multiple S-S bridges then these will not be broken, and alkylated.

Of course, if you're using LC-MS then poorly selected instrumental settings may also account for the low sequence coverage.

It might be helpful to simulate the digestion of your protein with trypsin on the available algorithm (https://www.expasy.org/resources/peptidemass) at the SWISS Institute, to give you an idea on whether the issue is with your protocol or the protein.

Hope this helps,

Ana

Thank you Ma'am for lightening response !

My protein of interest is having 175 Amino acids (molecular weight of 18kDa) if I do trypsin digestion there will be one big peptide from A.A 74-179 (attached image for same through in silico tool ) which will be undigested. So coverage as as result is less, that is why I was thinking to cut this bigger peptide left over after trypsin digestion using another Glu-C enzyme ! But I had doubt regarding GluC generating fragments of trypsin and vice versa.