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Squirrel Tutorial Pre – Pre-Process Steps Modify Single Ion (SI) M/z Calibration Fitting

Squirrel Tutorial Pre – Pre-Process Steps Modify Single Ion (SI) M/z Calibration Fitting Baseline Fitting. Donna Sueper. Aerodyne, University of Colorado, Boulder. Pre – Pre-Process Steps Tutorial Outline. Squirrel Quick-Look Flow Chart Review Squirrel Complete Analysis Flow Chart

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Squirrel Tutorial Pre – Pre-Process Steps Modify Single Ion (SI) M/z Calibration Fitting

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  1. Squirrel Tutorial • Pre – Pre-Process Steps • Modify Single Ion (SI) • M/z Calibration Fitting • Baseline Fitting Donna Sueper Aerodyne, University of Colorado, Boulder

  2. Pre – Pre-Process StepsTutorial Outline Squirrel Quick-Look Flow Chart Review Squirrel Complete Analysis Flow Chart MS Stick Calculation discussion, Screen Shots • modify SI (Results = 1 wave) • m/z fitting (Results = 3 waves) • baseline fitting (Results = 10 parameters)

  3. Quick-Look Analysis Flow Chart Get Index HDF Index Tab Pre-Process _p data (DAQ sticks) time series, avg spectrum image, avg size dist, size binned spectrum MS Tab PToF Tab

  4. Squirrel Analysis Flow Charts Quick Look Complete Analysis Pre-Pre-process steps Get Index Get Index Check m/z calibration Check baseline Modify SI HDF Index Tab HDF Index Tab m/z fitting params baseline fitting params Ion_user Pre-Process Pre-Process _p data (DAQ sticks) _p data (recalc sticks) time series avgs, etc. size dist, mass spec, image, etc. time series avgs, etc. size dist, mass spec, image, etc. MS Tab MS Tab PToF Tab Airbeam PToF Tab corr_fact Corrections Tab Do Corrections PToF _p data (dc marker) Calc Loadings Tweak Frag table Loading waves Frag Checks Tab Diagnostics Plot

  5. Complete Analysis > Modify SI (Single Ion) This step is only necessary if the values as entered & saved in the data acquisition software are wrong. This step can be done before or after the m/z calibration and baseline routines. On the HDF tab, press the Modify SI button. Change the SI values to correct values. The Single Ion value is used in converting units from bits*ns to Hz. If this wave exists, it will be used in the pre-process step. Example of how one would systematically change the SI values for all V or all W mode runs: Run this from the command line: root:diagnostics:ionSingleStr = 11.5*root:diagnostics:TofTypeVKey + 22*root:diagnostics:TofTypeWKey Modify SI Results (1 wave): root:diagnostics:ionSingleStr

  6. Complete Analysis > Check m/z Calibration On the HDF tab, press the Check m/z calibration button. View fit info for many runs Choose which peaks to fit View peak fits for individual runs Filter out bad runs

  7. Check m/z Calibration Discussion We need to map the data points from the acquisition software, which is measured in time, to m/z units. The formula is ion time of flight = intercept+slope*(mass^power) This is also stated as mass = ( (time – intercept)/slope ) ^(1/power) The data acquisition software uses 3 masses, sets the power parameter to 0.5 and saves the intercept and slope values for each run. Because it is imperative that we get this mapping correct, we need a scheme for calculating these 3 parameters using more than 3 peaks and a variable number of points for fitting each peak.

  8. Check m/z Calibration Panel (left side) Review the default choices for m/z peaks. Select an arbitrary run and the gold Show Run Values button. Observe the fits for individual masses. Repeat for other runs. • Adjust entries in table to get: • Well defined peaks • Low ppm values for accuracy • Yellow lines (fit at all chosen masses) near peak centers • Similar results for several runs

  9. Check m/z Calibration Panel (right side) Press Begin Peak Fitting button on left hand side. View right side graphs. After all runs complete you may do either/all: View individual runs (see previous slide) Filter bad values . View graph, table.

  10. Check m/z Calibration Panel (right side) Math details For individual masses: AccuracyMassm = ( ( mz as by fitm - mz_Massm ) /mz_Massm ) * 1e6 For the set of selected masses at one run: OneRun_deltaTSquared = ( (AllRunsMassSigma/sqrt(2)) * (2*sqrt(2*ln(2))) )^2 curveFit/n/q line OneRun_deltaTSquared /X=mz_Mass deltaTSquared_Chisq[currentRunIndex] = V_chisq/100 from line fitf ppmGuidelines = .1*(1/Mz)*sqrt(W_coef[0] + W_coef[1]*Mz) t_ion = sqrt(W_coef[0]) // W_coef from line fit Resolution= 1/ (2*(1/mz_FitSlopeVar)* sqrt(W_coef[1]) )

  11. Check m/z Calibration Panel m/z Calibration Results (3 waves): root:mzFitting:mz_FitSlope root:mzFitting:mz_FitIntercept root:mzFitting:mz_FitPower Before pressing End button or closing the window, make sure that you have no nan values in the 3 waves (for the runs you are analyzing).

  12. Complete Analysis >Check Baseline On the HDF tab, press the Check baseline button. Choose fitting parameters View many runs Choose stick compliment parameters View details of one run

  13. Baseline & Stick Calculation Discussion What do we need to calculate sticks? Stick = Area of peak integration region (less baseline) We need 3 items: (1) peak center (2) peak delta (3) baseline algorithm (1) Peak center determined by: integer amu (also what data acquisition software uses) OR user defined mass defect wave (2) Peak delta determined by: ‘resolution’ function

  14. Baseline & Stick Calculation Discussion Peak Delta -> Resolution formula: R0 * (1- ( 1/(1+exp((mass - m0)/dm) ) Resolution parameters control integration widths For c mode data default parameters are: R0=300, m0=30, dm = 50

  15. Baseline & Stick Calculation Discussion Peak delta formula: (integer mass)/(2*Resolution(integer mass) ) Example: At mass 100 for c-mode spectra using default resolution parameters, the peak delta width in amu is = 100/(2*Resolution(100)) = 100/(2*(300*1-(1/(1+exp((100-30)/50))))) = 0.207766 amu. Thus peak integration region is 0.415532 amu.

  16. Baseline & Stick Calculation Discussion We do not want any stick integration region to be >1. Otherwise, our peaks overlap. We need to define a maximum stick integration region. The default is .8 amu. This can be changed by the user. The mass value is displayed where the stick integration region is > .8 amu. This value serves only as a guide. For each mass, the peak integration region is the minimum of two values: (a) The peak integration region as found by the resolution formula and (b) the maximum stick integration region.

  17. Baseline & Stick Calculation Discussion (3) Baseline Stick complement is everything outside the peak integration regions. We will find & use the baselines of open spectra and the baselines of closed spectra, NOT the baseline of the difference spectra. Difference Spectra = (Raw Open – Baseline of Raw Open) - (Raw Closed – Baseline of Raw Closed) NOT (Raw Open – Raw Closed) – (Baseline of (Raw Open – Raw Closed) )

  18. Check Baseline Panel Our first task is to define the stick integration region (and thus the stick complement region). We examine the results for one run using default settings. Make sure the ‘Use new m/z calibration …’ is checked, choose a run number, then press the gold Show stick spectra button. View stick complement graphs.

  19. Check Baseline Panel Tweak resolution parameters, and perhaps the max stick region parameter. Press gold ‘Show spectra…’ button again. View stick complement graphs. Repeat with various runs.

  20. Check Baseline Panel One can tweak the peak integration regions by other options. Choose the MoreOptions tab. The stick complement region is smoothed before a fit is performed. If data from one run is too noisy, one can view the rough average of raw open, closed and difference spectra. The rough average is a simple point-by-point average and does not consider any slight m/z calibration shifts. This is for baseline panel display only – this option is not executed when recalculating sticks. See options in the Display Option tab. If you change options, press the gold ‘Show spectra…’ button again.

  21. Check Baseline Panel One can tweak the peak integration regions with more options. The stick complement region is smoothed before a fit is performed. If you check a box, new subpanels appear. If you change options, press the gold ‘Show spectra…’ button again. Use this option to remove additional regions from the stick complement. Enter 0 in the No-Man’s Land column for stick complement removal only (typical), and 1 to also remove this region when calculating sticks. Use this option to define the peak center to be non-integers.

  22. Check Baseline Panel The second task is to create a baseline, a fit of the stick complement. Choose baseline fitting type. If using a spline fit, press the ‘Estimate …’ button. Or enter your own values. Press calculate button. View fits of stick complement. Play with all the settings.

  23. Check Baseline Panel Baseline Results (10 parameters): Stick and baseline parameters that are NOT run # dependent! Before pressing End button or closing the window, make sure that your stick and baseline parameters are set to values of your liking.

  24. Check Baseline Panel Baseline Results (8 or 10 parameters): These 10 Parameters are the same for the entire experiment. 1, possibly 3 more 4 3

  25. Squirrel Analysis Flow Charts Quick-Look Complete Analysis Get Index Get Index Check m/z calibration Check baseline Modify SI HDF Index Tab HDF Index Tab m/z fitting params baseline fitting params Ion_user Pre-Process Pre-Process _p data (DAQ sticks) _p data (recalc sticks) time series avgs, etc. size dist, mass spec, image, etc. time series avgs, etc. size dist, mass spec, image, etc. MS Tab MS Tab PToF Tab PToF Tab The results from the pre-pre-process steps may be used when recalculatingsticks.

  26. Squirrel Analysis Flow Charts Quick-Look Complete Analysis Get Index Get Index Check m/z calibration Check baseline Modify SI HDF Index Tab HDF Index Tab m/z fitting params baseline fitting params Ion_user Pre-Process Pre-Process _p data (DAQ sticks) _p data (recalc sticks) MSSDiff_p 2-D matrix sticks, Hz time series avgs, etc. size dist, mass spec, image, etc. time series avgs, etc. size dist, mass spec, image, etc. MS Tab MS Tab PToF Tab PToF Tab

  27. Pre – Pre-Process Completion: Once we have pre-processed MS (and/or PToF sticks) we have a matrix of integrated signal of difference spectra. This stick matrix will essentially remain unchanged after the pre-process step. We use this matrix to generate time series or average mass spectra. Subsequent analysis steps deal with corrections (i.e. AB correction) and customizations (i.e. Frag table adjustments) based on this stick data.

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