Thermo RAW files are organized in two folders - one for all positive mode raw files and another for all negative mode raw files. These files will be loaded into MS-DIAL in two separate searches. Download MSP library files for Positive and Negative mode and save them in a folder in the computer you are using to perform the searches.

Open MS-DIAL

Click File -> New project

Project file path: Browse the folder with Positive or Negative raw files

Ionization type: Soft ionization (LC-MSMS)

Separation type: Chromatography (LC)

MS method type: Conventional LC/MS or data dependent MS/MS

Data type: MS1 Profile, MS/MS Centroid

Ion mode: Positive or Negative

Target omics: Lipidomics

Click "Next"

Analysis file path: browse to the folder with raw files, select all the apply for the search. Should include at least 3 blanks and 3 QC runs.

Choose the file type for each sample (Blank, QC, sample)

Class ID - add short group names for each replicate without adding numbers among replicates.

Data collection tab

Mass accuracy: MS1 tolerance 0.01 Da,

MS2 tolerance 0.025 Da

• Advanced:

Data collection parameters: retention time begin 0 min, retention time end 33 min, MS1 range begin 150 Da, MS1 range end 2000 Da, MSMS range begin 150 Da, MSMS range end 2000 Da, Isotope recognition: max charge number 2.

Peak Detection tab

Peak detection parameters: minimum peak height 10000 amplitude, mass slice 0.1 Da

• Advanced:

Smoothing method: linear weighted moving average

Smoothing level: 3 scan

Minimum peak width: 5 scan

MS2Dec tab

Deconvolution parameters: sigma window value 0.5,

MS2 abundance cut off 10 amplitude.

• Advanced:

Check Exclude after precursor ion Keep the isotope ions until 0.5 Da

Check Keep the isotopic ions w/o MS2Dec

Identification tab

MSP file and MS/MS identification setting: the library will be loaded automatically

Retention time tolerance: 100 min,

Accurate mass tolerance (MS1) 0.01 Da,

Accurate mass tolerance MS/MS 0.05,

Identification score cut off 80%.

Adduct tab

For positive mode acquisition:

Check [M+H]+

Check [M+NH4]+

For negative mode acquisition:

Check [M-H]-

Check [M-H2O-H]-

Alignment tab

Alignment parameters settings: Under result name add a name for the alignment file, reference file choose one of the QC runs, retention time tolerance 0.05 min, MS1 tolerance 0.015 Da

• Advanced:

Check remove feature based on blank information

Check keep "reference matched" metabolite features

Check keep ''suggested (w/o MS2)" metabolite features

Check keep removable features and assign the tag

Check Gap filling by compulsion

At the bottom

Check together with Alignment

Click "Finish" --> processing will start.

Once the search is finished, click on the name of the file that is showing at the Alignment Navigator

Click Export - Alignment result, choose a folder to save the file, check remove features from blank, and export.

Data can be loaded into Excel/R or any other software environment for further data analysis.

Data Analysis:

load data into Excel, filter out: m/z match FALSE and identifications named w/o MS2.

Combine positive and negative identifications

Summarize the data by lipid name (ex: using R), save as txt file.

Load the summarized into R or the online MetaboAnalyst 6.0 tool.

MetaboAnalyst 6.0 --> statistical analysis

Upload .txt data as peak intensities (check file format), no data filtering,

Sample normalization: normalization by median,

Data transformation: Log transformation (base 10), no Data Scaling.

At this point there are distinct analyses that can be done, our core provides the following as a first-step analysis:

Chemometrics analysis: PCA, PLS-DA with importance measures using VIP.

Univariate analysis: fold change and t-test -> volcano plot (two groups comparisons).

Clustering analysis: heatmap.