Hello, I recently heard about Skyline after ASMS 23 (everyone was using it!) and I have a few questions! I'm fairly new to the mass spec community and currently use compound discoverer/tracefinder for processing small molecule data for the metabolomics core I work in. These softwares are a bit more new-user friendly (AKA faster for me to be trained on/put to work on my own) but now that I know a little more about mass spec, I'm trying to find more efficient ways to process my data as we have over 250 targeted metabolites we screen per sample and have 100+ samples weekly. I'm wondering if I'm using Skyline correctly, as I find myself still having to manually go through each molecule and ensure the correct RT is chosen for all my samples (replicates).

Here is the current workflow I have been using:

1. Import small molecule transition lists (list of 250+ molecules) with RT, m/z values, adducts, and formulas
   Transition Settings are on Full scan; Count/Orbitrap/70,000ResolvingPower at 400m/z /PRM/Orbitrap/70,000ResolvingPower at 400m/z/Include all matching scans
   Molecule Settings are all on default, I do have a question about libraries I will ask below, and as for labels I only have heavy checked (I'm still not very clear on if I should have both or neither checked? I think heavy checked was the default for small molecules.
2. Import Results (samples)
3. Expand all precursors and ensure the correct peak is being integrated for each molecule (each molecule only has one precursor) while using the Synchronized Integration for all replicates.
   RT shift does not usually occur from replicate to replicate but does happen from batch to batch (ex: data processed from samples injected on Monday will likely have a small RT shift ~0.2 min compared to samples injected on Friday after 100+ samples have been injected). Is there any way to adjust this globally? Such as, shift all molecules RT +/- 0.2min? As of now I manually check the peaks for a few compounds, note the RT shift, and adjust the RT values on excel then reimport the transition list with the "new" shifted RT.
4. Export the molecule name, replicate name, and area (MS1 total area)
   These are the only features I will be needing from all the data I process as we have an internal pipeline we analyze it with. I have a standard that is injected separately as a sample that I use to ensure the correct peaks are being integrated. Is there any way to align all the molecules RT and integration to the standard if it is set as a sample on its own (a replicate)?

This workflow is repeated each time I make a new batch, is this the correct way to be working (importing the Transition List each time)? Is there a way to save this workflow/method/save the manual changes I have made to the molecules? Ex: I change the integration range but have no way of keeping that range when importing the transition list at the start of a new batch. I have been trying to get around this is by importing peak boundaries from previous batches, but still, as mentioned before, my RT shifts between batches so this isn't always useful to do.

Regarding libraries, I have made a spectral library using mzVault but am unsure how to import that into Skyline/if that is possible or if I need to use another software for creating the library/changing the format of the mzVault library file.

Sorry for all the questions! Please let me know any tips/help you have, I appreciate all of it and am excited to learn more about Skyline! :)

Sara