Hei Eric

There is definitely a flaw in your workflow. As you describe, it is inclear wheather it is "protein extraction", sample prep (digestion), instrument (method and mass accuracy) or analysis (database search related).

Andreas pointed you already to some relevant questions.

more questions are e.g:

What is your sample all about? (enough material?)

What are your FUSION methods (activation for msms)

What are your search parameters?

Any un-anticipated variable modifications eventually introduce upon sample prep?

(over-carbamidomethylation?, carbamylation?, deamidation, pyro-glu?)

In a GeLC-MS/MS approach you can for sure expect more than 2000 proteins (if material is not limiting) on the FUSION. (we get this number often already in a single shot). Limiting your protein list to minimum 2 peptides per protein, this usually cuts the list by about 30% while your confidence greatly improves.

There is a free software "Preview" by proteinmetrics which allows you to assess some of the described issues.. such as mass accuracy of ms1 and ms2. digestion statistics, open modification search!

-> give it a try.

best regards - jonas