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Accelerating Research in Life Sciences

Accelerating Research in Life Sciences. PREMIER Biosoft. Workflow for 2-AB labeled Bovine Fetuin N-Glycan analysis. Topics to be explained. New Project creation Loading Thermo Scientific native .raw file format LC-MS peak detection and picking

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Accelerating Research in Life Sciences

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  1. Accelerating Research in Life Sciences PREMIERBiosoft

  2. Workflow for 2-AB labeled Bovine Fetuin N-Glycan analysis

  3. Topics to be explained • New Project creation • Loading Thermo Scientific native .raw file format • LC-MS peak detection and picking • High Throughput glycan identification for the detected peaks • Portable report generation

  4. Launch SimGlycan and create a new project To create a new project, go to: File> New Project

  5. Enter a Project Name and press Create

  6. Import Thermo Scientific .raw data file To import Thermo Scientific .raw file, go to: File> Open> MS/MS Data File> Thermo Scientific Data File

  7. Navigate SimGlycan to the file location, select the file(s) and press Open Multiple data files can also be selected for loading

  8. Microsoft .NET Framework version 4.51 or higher is required To load Thermo Scientific .raw file, you will need Microsoft .NET Framework version 4.51 or higher installed on the computer. Press Help to know more.

  9. Load Scan window appears listing all MS and MS/MS spectra

  10. To load all scans, press Select All 1. Press Select All 2. All spectra are selected 3. Press Load Scan

  11. Press Specify Range to selectively load the spectra

  12. Upon pressing Load Scan, data loading starts

  13. Once completed, loaded data file appears under Profile node

  14. Click on LC-MS and MS/MS Data node

  15. Initiate LC-MS peak Detection To initiate LC-MS peak detection, go to: Analyze> LC-MS & MS/NS Data Pre-processing> Peak Detection and Picking

  16. Peak Detection and Picking window appears

  17. Select the Data file for peak picking 1. Select the Data File 2. Press the right arrow button

  18. Data file is now available under Selected Profile column Data File is available under Selected Profile column

  19. Specify Adduct combinations 1. Tick Select Adduct check box 2. Press Select Adduct button 3. Adduct Identification window appears. Specify the adduct combinations

  20. Check Template Name box and specify instrument specific peak picking template 1. Tick the check box Template Name 2. Specify Instrument or Workflow specific peak picking template

  21. Else, specify your own set of peak picking parameters Specify peak picking parameters Press Settings for advance parameters

  22. Specify advance peak picking parameters and press OK

  23. Press Preferred List to create a peaklist of your choice

  24. SimGlycan creates the peaklist

  25. Press View Chromatogram to view the XIC of a compound 1. Click on the compound specific row 2. Press View Chromatogram button 3. XIC of the selected compound appears

  26. Initiate High Throughput glycan search To initiate high throughput glycan search, go to: Analyze> LC-MS & MS/MS Data Pre-processing> High Throughput Glycan Search> Released Glycan and Glycopeptide Analysis

  27. Specify the Peaklist and press OK

  28. Specify the glycan search parameters

  29. Press Fragmentation Pattern

  30. Press Filter for setting filters to reduce false positives

  31. Select MS/MS spectra for glycan search 1. Press Select All 2. Press Add

  32. MS/MS spectra are selected for glycan search MS/MS spectra selected Press Search

  33. SimGlycan assigns an High Throughput Search ID. Press OK

  34. Check Search Status To check the search status, go to: View> HTP/GpQA Search Status

  35. SimGlycan displays the search status

  36. Retrieve the search results To retrieve the search results, go to: View> Fetch High Throughput Results> Load HTP Request

  37. Identified glycan are listed within Search Results tab

  38. View Glycan Structure 1. Select the compound specific row 2. Press Glycan Structure button 3. SimGlycan displays the glycan structure

  39. View Theoretical fragments 1. Select the compound specific row 2. Press Glycan Fragments button 3. SimGlycan displays the list of Theoretical fragments

  40. Press Annotated Peaklist see matched and unmatched fragments Blues are matched fragments Yellow are unmatched fragments

  41. Generate Mass Spectrum for Annotated peaks Press Plot Spectrum

  42. Specify Plot Type, Fragment details, and press OK

  43. Successive Fragment Loss plot

  44. Symbolic Representation plot

  45. Domon Costello Nomenclature plot

  46. Generate an Excel report of identified glycans 1. Press Generate Report within Search Results tab 2. Generate Report window appears. Select the parameters to be exported and press Ok

  47. Identified glycans, along with their structures are exported

  48. Thank You!

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