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E. Coli Spike-in Experiment

E. Coli Spike-in Experiment. Use Case/Training. LC/MS and GC/MS data processing workflow. .WIFF files (TOF) .D files (QTOF). Mass Hunter. MHD Files. LC/MS TOF. GeneSpring MS. GC/MS. AMDIS DB search (NIST). ELU/FIN files. .D files. GeneSpring MS.

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E. Coli Spike-in Experiment

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  1. E. Coli Spike-in Experiment Use Case/Training

  2. LC/MS and GC/MS data processing workflow .WIFF files (TOF) .D files (QTOF) Mass Hunter MHD Files LC/MS TOF GeneSpring MS GC/MS AMDIS DB search (NIST) ELU/FIN files .D files GeneSpring MS

  3. Installation of MassHunterRequired for converting WIFF files into MHD files • MassHunter requires .NET version 2.0 • http://www.microsoft.com/downloads/details.aspx?familyid=0856eacb-4362-4b0d-8edd-aab15c5e04f5&displaylang=en • MassHunter requires “AnalystQS” to be installed • Most Agilent and ABI TOF workstations already have this • Installer for AnylstQS is provided on DVD • F:\INSTALLATION\AnalystQS\Setup.exe • “Install” MassHunter by simply copying the complete folder “MassHunter (MFE31)” to your local hard disk

  4. Installation of GeneSpring MS 1.0 BETA • Need at least 1 GB of memory, 2 GB is recommended • For training, need at least 2 GB of disk space • MHD files are ~50-100 MB a piece

  5. Installation of GeneSpring MS 1.0 BETA • Run installer – setupGeneSpringMS-1.0.xx-win32.exe • Run GeneSpring MS 1.0 • Copy/Paste the contents of the file “LicenseKey.txt” into Enter License window

  6. Installation of GeneSpring MS 1.0 BETA • GeneSpring Splash Screen

  7. Start GeneSpring – First HurdleChoose “MassProfiler Example Data” as starting exp.

  8. Experiment Group opens up in Main GeneSpring Window

  9. Changing the Memory UseMenu Edit -> Preferences…

  10. Changing the Color Scheme

  11. TOF Experimental design • E. coli protein abstract spiked in with two Bovine proteins • Serotransferin • BSA • 18 Samples: • Three different concentrations, 3 injections, two different days 1100 HPLC + TOF MS

  12. Mass Hunter, the Feature Extractor of the MS world • Mass Hunter was used to extract the molecular features • Each peptide was “collapsed” from the multiply charged ions and isotopes into one measurement represented by a Rentention Time (RT) and a single, neutral mass • This functionality will be part of the MassHunter Workstation software for the QTOF, but current TOF users need to install MassHunter separately

  13. MassHunter analysis of the WIFF filesExtra Exercise

  14. MassHunter processes WIFF filesExtraction Parameters and Display Filters Most settings are fine in defaults, except for peptidic setting Use these if the sample is protein. Uncheck for metabolites

  15. Press Execute to process the WIFF files

  16. WIFF files directory will contain the MHD filesand some Excel files with similar information

  17. GeneSpring MS Main Window – Load MHD Drag-n-Drop very common UI in GeneSpring

  18. Import Data Files into a NEW Experiment Group

  19. Dialog allows for adding more files

  20. Alignment Parameter Settings – Controls which features are aligned

  21. Additional Filter Parameters to Restrict Features

  22. Results window of the alignment is RT vs Mass plot Number of masses found If everything looks OK, continue Allows for refining the alignment parameters

  23. New Experiment Group window appears

  24. Sample Attribute Window MIAPE compliant

  25. New Experiment Check listOnly first three are relevant (No error model in V 1.0)

  26. Normalization windowEdit the “Per Run: Normalize to 50th Percentile” step

  27. Set the “Use only measurements flagged” to“Anything but Absent”

  28. Default view of the results – Blocks view

  29. Switch to Graph View

  30. Your experiment’s a mess! 

  31. Setting up Parameters

  32. Experiment Parameters determine the “Grouping” of your samples

  33. We’ll add Four Parameters;Group, Day, BSA and Serotransferrin

  34. Adding a customer parameter

  35. Make BSA and Serotransferrin numeric and fmol as units

  36. Excel file “E. coli Spike-in – Experiment Parameters.xls”Copy paste into

  37. Parameters defined

  38. Setting up Interpretations

  39. Experiment Parameters in the “How to Display Parameters” Window

  40. Experimental setup – Group and Day determine thisSet Group and Day as Non-continuous parameters

  41. Group replicate injections by selecting Group as Continuous and Day as Non-continuous Parameter

  42. Switch between the two views by selecting the Interpretation in the Navigator

  43. Group all replicates together by selecting Group as the only continuous parameter

  44. Replicates from both days averaged

  45. More fun with InterpretationsPlot BSA or Serotransferrin concentration vs. Expression

  46. QCPCA on Conditions

  47. PCA on ConditionsChoose ALL SAMPLES interpretation

  48. Results of PCAPlot of each of the components loadings,

  49. Color by GroupDisplay Options

  50. Each block corresponds to one SampleGroup C similar to Group A

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