Squirrel Tutorial Pre – Pre-Process Steps Modify Single Ion (SI) M/z Calibration Fitting Baseline Fitting Donna Sueper ToF AMS Training, Aerodyne, Nov/Dec 2006 Aerodyne, University of Colorado, Boulder

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

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

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

Complete Analysis > Modify SI (Single Ion) On the HDF tab, press the Modify SI button. Modify SI Results (1 wave): root:diagnostics:ionSingleStr Change the SI values to correct values. 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. 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.

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

Check m/z Calibration Discussion We need to map the data points from the acquisition software, which is measured in time, to amu units. The data acquisition software uses 3 peaks (masses), sets the power parameter to 0.5 and saves the intercept and slope values for each run. The formula is ion time of flight = intercept+slope*(mass^power) This is also stated as Mass = ( (time – intercept)/slope ) ^(1/power) 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.

Check m/z Calibration Panel (left side) 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 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.

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

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.

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

Baseline & Stick Calculation Discussion (1) Peak center determined by: integer amu (also what data acquisition software uses) OR user defined mass defect wave 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 (2) Peak delta determined by: ‘resolution’ function

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

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))))) = amu. Thus peak integration region is amu.

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.

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) )

Make sure the ‘Use new m/z calibration …’ is checked, choose a run number, then press the gold Show stick spectra button. Check Baseline Panel View stick complement graphs. 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.

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

Choose the Options tab. Check Baseline Panel One can tweak the peak integration regions by other options. 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. If you change options, press the gold ‘Show spectra…’ button again.

If you check a box, new subpanels appear. If you change options, press the gold ‘Show spectra…’ button again. Check Baseline Panel Choose the Advanced Options tab. One can tweak the peak integration regions by even more options. Use this option to define the peak center to be non-integers. 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.

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

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

Check Baseline Panel Baseline Results (10 parameters): These 10 Parameters are the same for all runs.

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