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In this help topic, you will learn how to build a method using the PPL ReSpect deisotoping algorithm. To perform deisotoping, you need to have isotopically resolved MS data. If you do not have isotopically resolved data, you should use either ProMass charge deconvolution or PPL charge deconvolution methods. Once you have built a PPL method, the method can be embedded into a ProMass parameter file to enable automated deisotoping of entire LCMS data sets within the ProMass workflow. The example demonstrated in this help topic is oligonucleotides, but the same process is applicable to peptides, proteins, or any other type of sample. The basic steps for building a PPL deisotoping method include:
1.Import a representative ESI mass spectrum for your desired analyte into the PPL ReView program
2.Enter the ReView guided processing mode
3.Perform baseline correction of the data
4.Generate a model of the raw data based on the peak shape
5.Using the peak shape model, deconvolve the data and generate a peak list
6.Optionally filter the peak list to remove insignificant peaks
7.Enter a template molecular formula and deisotoping parameters
8.Produce a deisotoped result for your test data set
9.Save your PPL method file
10.Save your peak table file
Once you have built and saved your PPL method, you will want to add the PPL method to your ProMass parameter file, and set relevant ProMass parameters for deisotoping if you want to use the PPL method for automated deisotoping. Once you have added a PPL method to your ProMass parameter file you can utilize the PPL Auto Mode feature to adjust certain PPL parameters based on mass ranges, input sequence, and/or molecular formula.
Steps 1-3: Import a spectrum into ReView and enter guided processing mode
•Export a clipboard exact mass spectrum from Qual Browser, as described in a separate help topic. Note: Use only profile data. PPL software cannot operate on data that has already been centroided. The data shown in this help topic is the test24mer_std.raw file included with ProMass HR. The file is located in the directory: C:\Program Files\ProMassXcali\TestData\PPL deiso
•From the PPL tab in the ProMass Build Params utility, click the Send Clipboard to PPL ReView button.
•The spectrum should be displayed in the PPL ReView application.
•ReView will now enter the guided processing mode.
•The first step to be performed in guided processing is baseline correction. Automatic baseline correction is selected by default.
•Click the Next button to continue.
•Tip: If you are unfamiliar with the PPL ReView application, access the Help menu within ReView to find out more about the operation and features of this program.
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Step 4: Generate a peak shape model of the raw data
•The next step in PPL processing is to define a peak model. PPL will use these settings to model the shape of all raw data peaks for peak deconvolution purposes. In guided processing mode, ReView will automatically generate a peak model for you based on your data set. In the example below, the blue trace is the modeled data and the green trace is the raw (baseline corrected) data.
•You can use the mouse to zoom in and check to see how accurate the peak model is that has been created for you. The parameters in the peak model box can be changed if the model is not satisfactory. A shape of “2” is perfectly Gaussian. A shape of “1” is Lorentzian. For resolved orbitrap data, the shape should be close to a value of 2. The width values are in number of data points. A typical orbitrap data set is 2 data points wide on the left and right.
•If you decide to change a setting, click the ReCalc button to see how the model is affected by your change. To revert back to an automatically calculated model, click the Model button.
•Click the Next button to proceed after you are satisfied with your modeling parameters.
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Step 5: Peak shape deconvolution
•The next step in PPL processing is to perform peak deconvolution using the peak shape model that was generated in Step 4.
•After creating the peak model, select the Deconvolve option from the Peak Detection settings box (A), then click Next.
•Select a degree of fit setting of “2” from the Deconvolution dialog box (B). Then click the Go button. The “2” value has been found to work well for most orbitrap data sets.
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Step 6: Select peak filtering options
•Before proceeding with deisotoping, click the Select Peaks button from the Post Processing dialog box (C).
•In the Peak Selection dialog (D), enter a minimum peak significance of 2 standard deviations, a noise rejection setting of 95% confidence, and the use relative intensities option. These settings work best for most orbitrap data. If you want to turn off peak filtering, click the All Peaks button.
•Click on OK to exit the Peak Selection dialog.
•Select the Deisotope option from the Post Processing dialog box (C) and click on OK.
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Step 7. Enter deisotoping parameters
•The next step in PPL deisotoping processing is to select the deisotoping parameters.
•The unit cell composition defines the approximate molecular formula for the type of molecule under study. In the example below, we have entered a composition that approximates a typical composition of a DNA 20-mer with equal A, C, T & G bases. The ratios of atoms to one another (not the absolute atom numbers) are important. Therefore, the unit cell settings for a DNA 20-mer will work equally well for a 10-mer or a 60-mer. The table below lists a few common 20-mer unit cell formulas useful for oligonucleotide analysis. Pay particular attention to chemical modifications that have unusual elements that may impact the isotope distribution (e.g., sulfur). If you are working with peptides and proteins, you can use the protein Averagine formula: C 4.9384 H 7.7583 N 1.3577 O 1.4773 S 0.0417.
•Refer to the table below for atom percentages for typical molecule types. These values may be used in the unit cell composition dialog box.
•The minimum and maximum charges are important, as this will determine the mass range of charge deconvoluted results. Values of min and max charges of 1 to 25 will cover most applications up to ~60 bases for oligonucleotides.
•Select a mass error that safely brackets the calibration error of your instrument. A value of 10 ppm for orbitrap data is a very safe setting.
•Be sure to select the Negative ions option if you are working with oligonucleotides, or any application where negative ionization mode was used.
•Use the other options settings shown, as they have been found to be optimum for most applications.
•Click on OK to deisotope your data set and display the result in ReView.
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Atom Percentages for Typical Molecules used in Deisotoping Applications
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Molecule Type
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C
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H
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N
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O
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S
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P
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DNA
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29.86
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37.67
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11.49
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18.07
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2.91
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RNA
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28.88
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35.87
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11.40
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20.97
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2.89
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2'O-Methyl RNA
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29.25
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38.44
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10.45
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19.22
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2.65
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Phosphorothioated DNA
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29.86
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37.67
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11.49
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15.16
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2.91
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2.91
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sgRNA (typical guide strand)
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28.83
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35.83
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11.31
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20.87
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0.18
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2.99
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Peptide (Averagine)
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31.71
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49.82
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8.72
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9.49
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0.27
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Step 8: View deisotoped results
•After deisotoping is completed, the deisotoped mass spectrum will be displayed in a new window, as shown below.
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•Masses can be labeled in either PPL window by clicking the label peaks button. .png)
•Results can be validated using the overlay feature. After clicking on a peak in the deisotoped pane, related peaks are highlighted and labeled with their charges in the original spectrum pane. Select the overlay button in the ESI spectrum pane. .png)
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•You can zoom the isotopic regions to inspect how well the deisotoped overlay (orange) agrees with the original data.
Step 9: Save your PPL method
•You must save the PPL method if you want to use it later for ProMass HR processing.
•To save your PPL method, access the File | Export Method… menu item from the deisotoped spectrum pane. The Save As dialog box will appear, as shown below.
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•It is suggested that you save your method using a descriptive name. In the example below we have named the method DNA_z1-25.METHOD to indicate that the method was optimized for DNA using a charge range of 1-25.
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• Additional information about using PPL ReView is available from the ReView Help menu items.
Step 10: Save your peak table file
•The PPL deconvolved peak table contains the final peak list and all processing steps performed to generate the peak list. It is very useful to save the peak table file in case the PPL method needs to be edited. A PPL deisotoping method cannot be recalled and edited directly. The peak table file must be opened in ReView and the deisotoping function must be run to allow editing of the deisotoping parameters. A new PPL method can be saved as a result of this process.
•From the PPL ReView ESI spectrum pane, select File | Save Peak Table...
Once you have built and saved your PPL method, you will want to add the PPL method to your ProMass parameter file, and set relevant ProMass parameters for deisotoping if you want to use the PPL method for automated deisotoping.
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