Hi Alexandra,
This must have come at a particularly busy time and it got by us without a response. In this case the error, which is telling you have accumulated over 5,000,000 transitions in 49,095 peptides on line 399 of your FASTA file, is, in fact, serving its purpose well, which is to warn you that the resulting document is not going to be useful.

Think about it. That's over 100 peptides per line of FASTA and over 100 transitions per peptide.

Skyline is not meant for this type of completely unconstrained searching based on only a FASTA file. You will need to perform some kind of pre-filtering to either restrict your targets or create a library of prior measured peptides for larger searches. You can look for things not in libraries, but not this broadly with any hope of success.

Thanks for posting your question and the error message screenshot. Apologies for not answering in a timely manner. I assume you have moved on, but in case anyone else is looking for an answer on the support board, I hope this will help to clarify.

--Brendan