Squirrel Tutorial Pre – Pre-Process Steps Modify Single Ion (SI) M/z Calibration Fitting Baseline...
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Transcript of Squirrel Tutorial Pre – Pre-Process Steps Modify Single Ion (SI) M/z Calibration Fitting Baseline...
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 ChartCalculation discussion, Screen Shotsmodify SI (Results = 1 wave) m/z fitting (Results = 3 waves)baseline fitting (Results = 10 parameters)
Get Index
Pre-ProcessH
DF
Ind
ex T
ab
PT
oF T
abtime series,avg spectrum
image,avg size dist,size binned spectrum
_p data (DAQ sticks)
MS
Tab
Quick-Look Analysis Flow Chart
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
Co
rre
ctio
ns
Ta
b
_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
Fra
g C
he
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Ta
bTweak
Frag table
Do Corrections
corr_fact
Calc Loadings
Airb
ea
mP
To
F
_p data (dc marker)
Loading waves DiagnosticsPlot
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 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.
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 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.
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_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.
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
}
} peak rightpeak left
peak center
peak delta
peak integration region
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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m/z
Resolution, original c-mode parameters Resolution, R0*2 Resolution, m0*2 Resolution, dm*2
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
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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Peak Integration R
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.
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
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 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.
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-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.