Lys-C digestion for TMT labelling

[karthikskamath]

I was wondering how critical it is to perform Lys-C digestion prior to trypsin digestion for generating peptides for TMT labelling. Has anyone in the forum tested efficiency of doing dual enzyme and stand alone trypsin digestion? Is there more Lysine miss cleavages when we perform stand alone trypsin digestion?

[zougman]

With the use of a thorough sample prep method, combination of the two enzymes (Lys-C + trypsin) does reduce the number of missed cleavages (compared to the only trypsin). I have tested it.

[karthikskamath]

Many thanks zougman. I was wondering to what extend the miss cleavages were when digested with Lys-C + trypsin compared to trypsin only?
I did do a test and the difference I observed was very minimal. However, I did not have enough technical replicates to make my claim.
My concern is, Lys-C is relatively expensive enzyme and necessitates additional time for digestion. Hence, If it did not make stark differences in digestion efficiency, I might as well skip it in case of relatively less complex samples. Any input in this matter will be hugely appreciated.

[zougman]

Again, the results are going to depend on the sample prep method you use. In my case - I work with complex samples, e.g. cell pellets or tissues, and extract all proteins with SDS right away. When the low % of the missed cleavages is required, I prefer using Lys-C and trypsin simultaneously and the only commercially available enzyme mix at this moment is from Promega. I use basic trapping digest with S-Trap (STrap) method which is, in my opinion, ideal for processing of complex samples. Now, the numbers: for the fast 2-hour digest I get about 30% (trypsin only) and 15% (enzyme mix) of the missed cleavages, for the overnight digest I get about 17% (trypsin only) and 8.5% (enzyme mix) of the missed cleavages. The lower % of the missed cleavages does not necessarily mean the perceptible increase in the number of protein id's, though )

[karthikskamath]

Many thanks for the response. Much appreciated.