Frequently, during peptide characterization, utilizing multiple digestions with different enzymes will be needed. Combining digests from multiple enzymes can be utilized to maximize sequence coverage, circumvent PTM-induced missed cleavages, and allow for alternate peptide MS2 data for validating PTM localizations. To analyze these mixed sample sets generated with different enzymes, additional MS/MS Id (Byonic) nodes can be added to the workflows and set to only search specific samples in the data set. The following steps detail this process:
1. After configuring the Instrument Parameters and Modifications sections of the workflow, create a copy of the Byonic node:
Repeat this process until you have as many nodes as digestion conditions
2. Either move the new Byonic node(s) to the left, or move the Quant (Byologic) node to the right until it is the last node in the sequence.
3. Set each node's digestion parameters to match each digestion condition.
4. Configure each node's General -> 'Samples' field to only search samples with it's desired digestion conditions. To do this we will be changing the field to include part of the file name unique to the desired samples bracketed with *. For example with the following two samples:
'Tryp' corresponds with the samples utilizing a trypsin digest and 'LysC' corresponds with the samples utilizing a Lys-C digest. In this example, we would change the 'Samples' field to *Tryp* and *LysC* respectively. For the final layout below:
NOTE: Make sure to NOT to change the 'Samples' field in the Quant node. It should remain as *