squirrel tutorial pre – pre-process steps modify single ion (si) m/z calibration fitting

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

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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 - PowerPoint PPT Presentation

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Page 1: 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 SueperAerodyne, University of Colorado, Boulder

Page 2: Squirrel Tutorial Pre – Pre-Process Steps  Modify Single Ion (SI)  M/z Calibration Fitting

Pre – Pre-Process Steps Tutorial Outline

Squirrel Quick-Look Flow Chart Review

Squirrel Complete Analysis Flow ChartMS Stick Calculation discussion, Screen Shotsmodify SI (Results = 1 wave) m/z fitting (Results = 3 waves)baseline fitting (Results = 10 parameters)

Page 3: Squirrel Tutorial Pre – Pre-Process Steps  Modify Single Ion (SI)  M/z Calibration Fitting

Get Index

Pre-ProcessH

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abtime series,avg spectrum

image,avg size dist,size binned spectrum

_p data (DAQ sticks)

MS

Tab

Quick-Look Analysis Flow Chart

Page 4: Squirrel Tutorial Pre – Pre-Process Steps  Modify Single Ion (SI)  M/z Calibration Fitting

Get Index

Pre-ProcessHD

F I

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Quick LookP

To

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abtime series

avgs, etc.

size dist,mass spec,image, etc.

_p data (DAQ sticks)

Complete Analysis

Pre-ProcessHD

F I

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Get Index

Check m/z calibration

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rre

ctio

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Ta

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_p data (recalc sticks)

m/z fitting params

MS

Ta

b

Check baseline

baseline fitting params

Squirrel Analysis Flow Charts

PT

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Ta

btime seriesavgs, etc.

size dist,mass spec,image, etc.

MS

Ta

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Modify SI

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bTweak

Frag table

Do Corrections

corr_fact

Calc Loadings

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_p data (dc marker)

Loading waves DiagnosticsPlot

Pre-Pre-process steps

Page 5: Squirrel Tutorial Pre – Pre-Process Steps  Modify Single Ion (SI)  M/z Calibration Fitting

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.

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

Page 6: Squirrel Tutorial Pre – Pre-Process Steps  Modify Single Ion (SI)  M/z Calibration Fitting

Complete Analysis > Check m/z Calibration

Choose whichpeaks to fit

View peak fitsfor individual runs

View fit infofor many runs

Filter out bad runs

On the HDF tab, press the Check m/z calibration button.

Page 7: Squirrel Tutorial Pre – Pre-Process Steps  Modify Single Ion (SI)  M/z Calibration Fitting

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 data acquisition software uses 3 masses, sets the power parameter to 0.5 and saves the intercept and slope values for each run.

The formula ision time of flight = intercept+slope*(mass^power)

This is also stated asmass = ( (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.

Page 8: Squirrel Tutorial Pre – Pre-Process Steps  Modify Single Ion (SI)  M/z Calibration Fitting

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.

Page 9: Squirrel Tutorial Pre – Pre-Process Steps  Modify Single Ion (SI)  M/z Calibration Fitting

Check m/z Calibration Panel (right side)

After all runs complete you may do either/all:

View individual runs (see previous slide)Filter bad values .View graph, table.

Press Begin Peak Fitting button on left hand side.

View right side graphs.

Page 10: Squirrel Tutorial Pre – Pre-Process Steps  Modify Single Ion (SI)  M/z Calibration Fitting

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]) )

Page 11: Squirrel Tutorial Pre – Pre-Process Steps  Modify Single Ion (SI)  M/z Calibration Fitting

Check m/z Calibration Panel

m/z Calibration Results (3 waves):root:mzFitting:mz_FitSloperoot:mzFitting:mz_FitInterceptroot: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).

Page 12: Squirrel Tutorial Pre – Pre-Process Steps  Modify Single Ion (SI)  M/z Calibration Fitting

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.

Page 13: Squirrel Tutorial Pre – Pre-Process Steps  Modify Single Ion (SI)  M/z Calibration Fitting

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

}

} peak rightpeak left

peak center

peak delta

peak integration region

Page 14: Squirrel Tutorial Pre – Pre-Process Steps  Modify Single Ion (SI)  M/z Calibration Fitting

Baseline & Stick Calculation Discussion

Peak Delta -> Resolution formula:R0 * (1- ( 1/(1+exp((mass - m0)/dm) )

Resolution parameters control integration widthsFor c mode data default parameters are:R0=300, m0=30, dm = 50

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Resolution, original c-mode parameters Resolution, R0*2 Resolution, m0*2 Resolution, dm*2

Page 15: Squirrel Tutorial Pre – Pre-Process Steps  Modify Single Ion (SI)  M/z Calibration Fitting

Baseline & Stick Calculation DiscussionPeak 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.

}

} peak right = 100.207766peak left = 99.7922

peak center = 100

peak delta = .207766

peak integration region = 0.415532

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Peak Integration R

egion, amu

Resolution, original c-mode parameters Resolution, R0*2 peak integration, amu peak integration, R0*2, amu

Page 16: Squirrel Tutorial Pre – Pre-Process Steps  Modify Single Ion (SI)  M/z Calibration Fitting

Baseline & Stick Calculation DiscussionWe 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. 600

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egion, amu

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.

Page 17: Squirrel Tutorial Pre – Pre-Process Steps  Modify Single Ion (SI)  M/z Calibration Fitting

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) )

stick complement

peak integration region

stick complement

baseline fit= interpolated stick complement

Page 18: Squirrel Tutorial Pre – Pre-Process Steps  Modify Single Ion (SI)  M/z Calibration Fitting

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.

Page 19: Squirrel Tutorial Pre – Pre-Process Steps  Modify Single Ion (SI)  M/z Calibration Fitting

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.

Page 20: Squirrel Tutorial Pre – Pre-Process Steps  Modify Single Ion (SI)  M/z Calibration Fitting

Choose the MoreOptions tab.

Check Baseline PanelOne 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.See options in the Display Option tab.

If you change options, press the gold ‘Show spectra…’ button again.

Page 21: Squirrel Tutorial Pre – Pre-Process Steps  Modify Single Ion (SI)  M/z Calibration Fitting

If you check a box, new subpanels appear.If you change options, press the gold ‘Show

spectra…’ button again.

Check Baseline PanelOne can tweak the peak integration regions with 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.

The stick complement region is smoothed before a fit is performed.

Page 22: Squirrel Tutorial Pre – Pre-Process Steps  Modify Single Ion (SI)  M/z Calibration Fitting

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.

Page 23: Squirrel Tutorial Pre – Pre-Process Steps  Modify Single Ion (SI)  M/z Calibration Fitting

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!

Page 24: Squirrel Tutorial Pre – Pre-Process Steps  Modify Single Ion (SI)  M/z Calibration Fitting

Check Baseline Panel

Baseline Results (8 or 10 parameters):These 10 Parameters are the same for the entire

experiment.

4

3

1, possibly 3 more

Page 25: Squirrel Tutorial Pre – Pre-Process Steps  Modify Single Ion (SI)  M/z Calibration Fitting

Get Index

Pre-ProcessHD

F I

nd

ex

Ta

b

Quick-LookP

To

F T

abtime series

avgs, etc.

size dist,mass spec,image, etc.

_p data (DAQ sticks)

Complete Analysis

Pre-ProcessHD

F I

nd

ex

Ta

b

Get Index

Check m/z calibration

_p data (recalc sticks)

m/z fitting params

MS

Ta

b

Check baseline

baseline fitting params

Squirrel Analysis Flow Charts

PT

oF

Ta

btime seriesavgs, etc.

size dist,mass spec,image, etc.

MS

Ta

b

Modify SI

Ion_user

The results from the pre-pre-process steps may be used when recalculating sticks.

Page 26: Squirrel Tutorial Pre – Pre-Process Steps  Modify Single Ion (SI)  M/z Calibration Fitting

Get Index

Pre-ProcessHD

F I

nd

ex

Ta

b

Quick-LookP

To

F T

abtime series

avgs, etc.

size dist,mass spec,image, etc.

_p data (DAQ sticks)

Complete Analysis

Pre-ProcessHD

F I

nd

ex

Ta

b

Get Index

Check m/z calibration

_p data (recalc sticks)

m/z fitting params

MS

Ta

b

Check baseline

baseline fitting params

Squirrel Analysis Flow Charts

PT

oF

Ta

btime seriesavgs, etc.

size dist,mass spec,image, etc.

MS

Ta

b

Modify SI

Ion_user

MSSDiff_p2-D matrix sticks, Hz

Page 27: Squirrel Tutorial Pre – Pre-Process Steps  Modify Single Ion (SI)  M/z Calibration Fitting

Pre – Pre-Process Completion:

Once we have pre-processed MS (and/or PToF sticks) we have a matrixof 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.