Accelerating Research in Life Sciences PREMIER Biosoft Accelerating Research in Life Sciences 1

A High Throughput Lipid Characterization Tool using HDMSE Data 2

Major Features Database containing 40,298 lipid structures and 15,09,305 structure-specific in-silico MS/MS characteristic ions. Load raw data from native files (.raw) for HDMSE workflows. Data processing – peak detection, peak picking – for LC-, SFC- HDMSE workflows. 3

Major Features Visualize raw data, and processed data – TIC, spectra, XIC of detected compound in retention time scale, XIC of compound in Drift Time scale, XICs overlay of precursor ion m/z, and its fragment ions in Drift Time scale. High throughput lipid structural identification using exact mass, and product ions from HDMSE workflows. Automated annotation of MS/MS spectra with IDed lipids, and their fragments. 4

Create a New Project 1. File > New > Project 5

Create a New Project 2. Enter a project name 3. Click Create 6

Import Data from Native as well as Standard Data Files 2. Select file type 1. File > Open > Peaklist Data > Waters Data File > Waters (DIA) > HDMSE (.raw) 7

Use the Shortcut to Import Waters Native File as Shown Below 1. Select Waters native file shortcut icon 8

Use the Shortcut to Import Waters Native File as Shown Below 1. Select Waters native file shortcut icon 2. Select the file(s) 3. Click Open 9

Specify Range 1. Select Profile Data 2. Press OK 10

Retrieving Selected Scan Number Information 11

The Selected Files/Scans can be Viewed from the Project Management Panel Along with Chromatogram and Mass Spectrum Loaded Data 12

The Selected Files/Scans can be Viewed from the Project Management Panel Along with Chromatogram and Mass Spectrum Tabular View Chromatogram view Loaded data selected Select row Search Results/ Mass Annotation & Lipid information are not applicable for DIA data Spectrum View 13

Chromatogram and Mass Spectrum Zoom Options 1. Pinned Chromatogram view 14

Chromatogram and Mass Spectrum Zoom Options 2. Select settings for zooming 3. Click OK 15

Chromatogram and Mass Spectrum Zoom Options Selecting Chromatogram area for zoom 16

Chromatogram and Mass Spectrum Zoom Options Pinned Spectrum View 17

Chromatogram and Mass Spectrum Zoom Options 2. Select settings for zooming 3. Click OK 18

Chromatogram and Mass Spectrum Zoom Options Selecting Spectrum area for zoom 19

Peak Detection and Picking 1. Analyze > DIA Peak Detection > RT + DT 2. Select Profiles 3. Select Template (HDMSe) 4. Select Settings 20

Peak Detection and Picking 1. RT Peaks 21

Peak Detection and Picking 2. Precursor Ions 22

Peak Detection and Picking 3. Product Ions 4. Press OK 23

Peak Detection and Picking 5. Select OK 24

Peak Detection and Picking 25

CID's are Detected and Displayed under LC-MS and MS/MS Data Node 26

High Throughput Lipid Search 1. Go to Analyze > LC-MS Pre-processing > High Throughput Lipid Search 3. Select MS/MS Data option to perform HTP search only for MS/MS data 2. Specify Range 4. Click OK 27

Specify HTP Search Parameters 1. Select profiles 2. Specify Search Parameters 5. Click Search 4. Click OK 3. Specify Filter Lipids 28

High Throughput Lipid Search 5. Click OK 29

A high throughput search ID number is generated HTP ID 30

Check HTP Status and Load Results Click this button to check HTP search status 31

Check HTP Status and Load Results HTP search status-Completed 32

Check HTP Status and Load Results Load HTP Results 33

Search results are displayed as shown below Search Results are displayed here 34

Lipid MAPS TG standard mix I (D5 labeled) Species # C:DB Neutral Formula NH4 adduct NL sn1+NH3 NL sn2+NH3 NL sn3+NH3 Rt 20:5/22:6/20:5 62:16TAG C65H89D5O6 993.77 674.57 648.57 14.21 20:4/18:2/20:4 58:10TAG C61H93D5O6 949.80 628.60 652.60 14.99 14:0/16:1/14:0* 44:1TAG C47H83D5O6 771.72 526.52 500.52 15.08 20:2/18:3/20:2 58:7TAG C61H99D5O6 955.85 630.65 660.65 15.54 15:0/18:1/15:0 48:1TAG C51H91D5O6 827.79 568.59 528.59 15.74 17:0/17:1/17:0 51:1TAG C54H97D5O6 869.83 582.63 584.63 16.15 16:0/18:0/16:0** 50:0TAG C53H97D5O6 857.83 556.63 16.31 19:0/12:0/19:0** 542.63 640.63 20:0/20:1/20:0 60:1TAG C63H115D5O6 995.97 666.77 668.77 17.19 *Not specified in the sample mixture but may be present as a contamination. **Isobaric species.  

Mass Spectrum Annotation Sorting by m/z value 36

Mass Spectrum Annotation 37

Mass Spectrum Annotation 38

Mass Spectrum Annotation 39

Mass Spectrum Annotation 40

Mass Spectrum Annotation 41

Mass Spectrum Annotation 42

Mass Spectrum Annotation 43

Mass Spectrum Annotation 44

Mass Spectrum Annotation 45

Mass Spectrum Annotation 46

Mass Spectrum Annotation 47

Mass Spectrum Annotation 48

Mass Spectrum Annotation 49

Mass Spectrum Annotation 50

Mass Spectrum Annotation 51

Mass Spectrum Annotation 52

Mass Spectrum Annotation 53

Mass Spectrum Annotation 54

Generate High Throughput Report 2. Select Profiles 1. File > Generate High Throughput Report 3. Click OK 4. Specify MS/MS Search Parameters 5. Specify the sorting options for organizing results in report 6. Specify the lipid information to be exported in report 7. Select output file type 8. Click OK 55

Results Exported into MS Excel File 56

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