Introduction. Isopro is an isotopic distribution simulator created to look at large biomolecules. Isopro was created using Microsoft Visual Basic and runs under Windows 3.1. Isopro will run on any 286 class computer, but a 386 or better is recommended for speed, especially for large empirical simulations. Some older (pre '91) 486 computers have a known problems with their floating point coprocessors, and might produce errors while performing resolution calculations. If you are still running a computer like this, buy a new one.
What's new? Isopro V 2.1 has a whole host of new features requested by over 1 million new users of the program. The GUI now provides more options and is less sloppy. It uses double precision math and the actual algorithm that has been thoroughly tested and generously provided by J.A. Yergey, but has been optimized for the PC. This means that you might be able to trust the results it produces (but again nothing is guaranteed), and are not restricted to C,H,N,O,S, and Fe. This version was created using VB 3.0, which means arrays are no longer restricted to 64 kB, so larger simulations with lower thresholds will not crash. On a 486-33, carbonic anhydrase (29 kDa) can be generated at the 0.1% level in about 40 seconds. Peak fitting is slower, but this is because more peaks are being generated at the same threshold. Plotting now also has user-definable limits. Listing has been changed to present the data in a more useful format. One last note, file formats have changed, so all old files are now fairly useless. Bug fixes: Peak lists for simulations with metallic elements now display properly. The algorithm has also been corrected to provide more accurate results for metallic elements.
Installation. Isopro can be installed by running the setup program included in the ISOTOPES.ZIP file. If Isopro does not run, make sure GRID.VBX and VBRUN300.DLL were copied to your windows\system directory and TABLE.TXT is in the working directory specified for ISOPRO.EXE.
Periodic Table. The start-up screen is now the periodic table. By clicking on an element, you can select the number of atoms to be included in the formula. The periodic table can be modified by making changes to the "table.txt" file in your working directory (see Isotopic Abundances).
Molecular Weights. Isopro will calculate molecular weights for the formula you have entered in the main window by selecting Molecular Weight from the Process menu. You have a choice of monoisotopic (most abundant isotope for each element) or average.
Simulation Parameters. Choosing Adjust Simulation under the Process menu allows adjustment of simulation parameters. Maximum Peaks is the maximum number of peaks allowed before the threshold is incremented. If the simulation goes beyond the maximum number of peaks specified, the threshold will be incremented by a factor of Threshold Increment and the peak list will be trimmed to fit this threshold. 10,000 peaks works well, but it may be increased for more accurate simulations, or decreased for computers with less memory.
Simulations. Isopro simulates isotopic abundances for molecules with any element in the periodic table, or any user definable element that can be created by modifying the "table.txt" file. There are two methods for simulation. When Exact is the chosen simulation type, Isopro will calculate the isotopic abundances using an algorithm previously published and provided by J.A. Yergey. The important parameter for exact calculations is the threshold value. The calculations will ignore any final abundance that is less than this value. Smaller thresholds will give more accurate results, but larger thresholds will be calculated faster. The calculations are quick enough that small thresholds do not impose a real problem. The system is limited to 30,000 peaks, so the threshold will automatically be increased when this limit is reached. Empirical simulations can be performed by selecting Empirical as the simulation type. The important parameter for empirical calculations is the number of simulations. To get a more accurate distribution, use a larger number of simulations. The empirical simulation works by randomly determining what isotope each atom in the molecule will be. It then combines the masses of each atom to get a molecular weight for that specific molecule. It then repeats this process the number of times that you have specified with the simulations parameter. Empirical simulations combine molecules with similar nominal masses together. To stop a simulation of either type before it finishes, press the cancel button.
Charge. Isopro will adjust the m/z according to the charge specified under the Charge selection of the Process menu. The assumption is made that a proton is the charge carrier. To reset the charge of an old simulation to a new charge, type in the new charge and click on the Reset m/z button. All new simulations will have initial charges of zero.
Plotting. When you choose the Plot option under the Action menu after completing a simulation, the abundance plot window will appear. No peaks will appear initially. Choosing Draw under the Process menu will display the spectrum according to display parameters set under the Properties menu. Choosing Peak under the Properties menu allows for the adjustment of Peak Type (Gaussian and Lorentzian) and Resolution (defined as FWHH). Display allows for the adjustment of the plot window (either automatic or user- defined) and the type of spectra to be displayed. Plotting for low threshold simulations with high resolution (especially lorentzian peak shape) is slow, but what do you want for free?
Listing. To list out the abundances for the simulation, choose the List option under the Process menu after you have completed your simulation. Isotopic combinations of the same nominal molecular weight are combined, with peak spread and multiplicity being reported. For exact simulations, the abundance is equal to the probability for that isotope combination. For empirical simulations, the abundance is equal to the number of molecules that had the specific nominal mass. If you would like to see the full listing for exact simulations, then save the simulation and view the file in a text editor. The list option is disabled for mixtures.
Printing. Hard copies can be obtained from either the plot or list of abundances by choosing Print under the Plot Process menu, or by clicking the print button in the list window. Output will be sent to the default Windows printer. It is recommended that you set up your printer for portrait mode for printouts of the list and landscape mode for printouts of the plots. This can be altered by choosing the control panel option under the file menu, and then choosing printers and making the necessary changes. Printouts of the plots follows WYSIWYG conventions and should look the same as what is currently in the graphics window, as long as you don't change any parameters before printing. To produce HPGL output for importing into a graphics program, add the HP plotter as a printer and set its port to FILE. When you click the PRINT button, you will be prompted for the file name you would like to print to. This file can then be imported into most graphics packages. The Windows print driver for HP printers has a bug, so plotting to file for very high resolution spectra may have problems (not my fault).
File Options. To save a simulation, choose Save under the File menu. Navigate through the drive and directory boxes so that your desired directory is displayed. Then either highlight the file you would like to store your results in, or type the file name in the text box, then click the Save button. To retrieve an earlier simulation, choose Open from the File menu. Again, navigate the drive and directory boxes so that you end up in your desired directory, and either highlight the input file, or type the file name in the text box, then click the Open button. To simulate a mixture, first simulate or load one component, then return to the File Open form, except click the Mixture button. To mix simulations, they must be of similar simulation types. Abundances for the component added with the Mixture button can be adjusted by changing the Concentration text.
Isotopic Abundances. Isotopic abundances and isotopic masses can be adjust by editing the "table.txt" file in the working directory. This is an ASCII file with the following format:
Number of Elements
Element_1_Symbol (limit two characters)
Element_1_Name (limit ten character)
#_of isotopes for Element_1
Isotope_A_Mass Isotope_A_Abundance
Isotope_B_Mass Isotope_B_Abundance (up to ten isotopes)
Element_2_Symbol ...
By modifying this table, isotopic abundances and masses can be adjusted. User-defined elements can also be created by substituting for a normal element.
Warning. The routines in this simulator have not been thoroughly tested, and the creator does not guarantee that anything will work properly. Also, this program has very little error checking, so if you attempt something strange, it will probably crash out and end up closing.
1. Yergey, James A., International Journal of Mass Spectrometry and Ion Physics, 52, (1983), 337-349.