edited: 31.7.2006 Perttu Haimi ( perttu.haimi@helsinki.fi
)
LIMSA (Lipid Mass Spectrum Analysis) is a program for quantitative analysis of mass spectra of complex lipid samples. LIMSA can do peak finding, integration, assigning, isotope correction and quantitation with internal standards. LIMSA integrates with Microsoft Excel and installs three new menu items: “Search lipids”,
“Batch analyze” and “Summarize results” to the Tools menu. The main user interface of LIMSA is opened by choosing “Search lipids” menu item.
Analyzing spectra
You need to copy and paste the mass spectrum to the first columns of the worksheet.
The first colum should contain the masses and the second column should contain the intensities. Parameters affecting the lipid analysis are organized into parameter groups
(see figure 1, marked with 1). Choose the appropriate parameter group and press
Search for starting the analysis. You can edit and save the changes you have made to the parameter group with either using the same name or with a new name,creating a new parameter group. A parameter group consists of a list of compounds expected to be in the spectrum (figure 1, marked with 2) and some parameters (figure 1, marked with 3) which control the analysis.
Parameters
Limsa supports three isotope correction algorithms. Subtraction method uses the integrated area of the first isotope peak and corrects for the isotope overlap by scaling and subtracting the calculated isotope pattern form subsequent peaks. Linear fit finds the best fit of calculated isotope patterns to the integrated peak areas. It uses all the isotope peaks, so it should give slightly more accurate results than the subtractive method, provided that there are no interfering compounds at the higher isotope peaks.
Peak model fit finds the best fit of gaussian peak patterns to the observed data. It is slower, but performs better if the resolution of the spectrum is poor.
Spectrum type can be either a spectrum, with multiple intensity measurements per peak, or a peak list. Peak model fitting cannot be used with peak lists. Spectrum can be either positive or negative mode, which affects the list of available compounds
(figure 1, marked with 4).
Specific fragment defines the instrument mode. If the field is left empy, LIMSA will calculate the isotope patterns as they appear in direct MS. If the fragment is given, the neutral loss or parent scan pattern is calculated. The calculation assumes that the first peak of the fragment is selected.
There are a few parameters affecting peak finding and integration. The offset can be used move the spectrum if the calibration of the mass spectrometer is a bit off.
Positive values move the spectrum to the left (makes the masses smaller), negative to the right. Peak fwhm is the full widht at half maximum of the peak. This parameter affects the peak finding, integration and assignment. The sensitivity parameter controls the peak finding, and is defined as the intensity of the peak divided by the largest peak in the spectrum.
Compoundlists

A compoundlist (fig 1, marked with 2) contains the compounds that you are expecting to find in the spectrum. The compounds can be grouped to classes, which affects the quantitation. The standards are used for quantitating other compounds in the same class. The standards are defined by giving them a concentration.
Output
The figure 2 shows the ouput of the program consists of 2) the peak list of found peaks, 3) the spectrum plotted with found peaks and assigned (fitted) peaks, 4) the residual of the fit, 5) the table of peak areas and concentrations and 6) the calibration curve

Compound library
Extended molecular formulas
You can use parentheses in in the formulas to group elements. If there is a number following the parentheses, all the elements inside the parentheses are multiplied by the number. So, for example (CH2)10 will equal C10H20. You can also use negative numbers, provided that the total number of no element in the formula becomes negative. For example, (H2)-1 could be used to indicate one double bond, (H2)-2 two double bonds and so forth. Custom patterns can be defined for example to use experimentally measured isotope patterns of partially labeled compounds, and their names must be enclosed in square brackets in the molecular formulas. For example
[Serine-2d] could be used to indicate a serine molecule with two deuteriums. Custom elements can be used as a part of larger molecules unless they are high resolution
(more than one peak per m/z value).
Editing the compound library
When adding new compound, you must give it a name, sumformula and charge. You can also make adducts by using the “insert selected compounds” -button. This will add the sumformulas and charges from the selected to the compound currently under construction.

the basis of the pattern calcula tion.
Curren tly,
LIMS
A has the list of the isotope abunda ncies of all the elements. You can define you own elements by adding them to the element list. The name can be arbitrary, but must not contain whitespace. The isotope pattern is constructed by adding peak at the time. The intensities of the peaks must be normalized so that they sum to one.
Miscel laneou s
Defini ng custom pattern s
Limsa uses predefined pattern s for

Importing and exporting
Using import and export functions you can copy the current parameter groups, compounds or patterns to an empty worksheet. This is useful for making backups or when sharing the data with others. Importing does not replace existing groups, compounds or patterns if they already exist in the database, so you must either to change the names or delete the old data.
Batch analysis
You can analyze a set of spectra using batch analysis, which lets you select the worksheets containing the spectra, and the parameters of the analysis. With summarize results you can copy the results from separate sheets to one worksheet.