My study is about cell metabolomics. I’m currently analyzing MS/MS data using MS-DIAL 5 for untargeted metabolomics, but I’m facing an issue where I don’t get any reference-matched/library-matched compounds (I got some, like 4 or 5, but they're not from metabolites of interest and seem like they're from contaminations). Instead, most of the annotated metabolites are either "suggested" or "w/o MS2" annotations. And some of the metabolites of interest regarding energy metabolism are also annotated with "w/o MS2". From what I understand, you cannot take w/o MS2 results with confidence.

Previously, I have also tried increasing the concentration and extracting more cells for metabolites but still face similar issues. I have also played around with MS-DIAL parameters, but still, I can't get the library matched.

My questions are:

1. Can w/o MS2 annotations be considered confident identifications and be used as results or in publication?

2. Are there specific parameter settings in MS-DIAL that I should check to ensure MS2 spectra are being used correctly for annotation?

3. Could this be the problem with the sample preparation or instruments/methods rather than data analysis?

Any advice on troubleshooting or optimizing settings of either MSDIAL or the instrument would be greatly appreciated!

My Setup:

• Instrument: Agilent 6520 Accurate-Mass Q-TOF LC-MS (Data Acquisition: Auto MS/MS or DDA)
• Sample: metabolite extracted from human cell lines
• File type: abf file using Abf File Converter / raw .d from Agilent (drag to MS-DIAL)
• MS-DIAL 5.0 settings: Default parameters with public libraries (MSMS_Public-all-pos-VS19/MSMS_Public-all-neg-VS19)

Thank you!