In order to start a single quantitation a raw file (Raw file:) and an Excel file (Quantitation) containing the mass list is required. The default settings for the input fields can be entered in the LipidDataAnalyzer.properties file (see Configuration settings).
Raw file: File in mzXML or chrom format is required. However, it is possible to use Thermo RAW format and the Waters Mass Lynx raw directories directly if XCalibur or MassLynx is installed.
Quant. files: Here the name of an Excel file has to be entered, containing the mass list. The format of the file is described in Preparation of the mass list Excel file.
Time before tolerance: This field specifies the retention time tolerance in minutes before the entered retention time in the Excel file. Example is given after “Time after tolerance”. This field is just used if a retention time has been entered in the mass-list Excel file.
Time after tolerance: This field specifies the retention time tolerance in minutes after the entered retention time in the Excel file. Example is given in the next paragraph. This field is just used if a retention time has been entered in the mass-list Excel file.
Example for time tolerance setting: E.g. in the excel file the retention time for a specific analyte is specified with 24 minutes, in the time before tolerance 2min and in the time after tolerance 3min is entered. Then, the algorithm will look for the analyte in the time range from 22-27 minutes.
Rel. base-peak cutoff: Peaks that are less intense than this per mille value of the highest identified are discarded by the algorithm. For the determination of the highest peak, just found analytes are taken into account (irrespective of the analyte class). This threshold can accelerate the quantitation, since intensities that are too small are discarded before the 3D quantitation is performed.
RT-shift: The retention time could shift from batch to batch. The entered value will be added to the retention time defined in the mass list file. Thus, the mass list file has not to be changed every time.
Isotopic quantitation of … isotopes where … isotopic peaks have to match: The checkbox indicates if in addition to the +0 peak other isotopic peaks should be quantified. The first value determines the amount of additional isotopes to be quantified (e.g. 2 would mean that +0, +1, and +2 isotopes will be quantified). The second value determines how many isotopes have to conform the theoretical isotopic distribution (e.g. 1: just the +1 peak has to conform).
Find molecules where the retention time is unknown: If this checkbox is selected, molecules in the mass list without retention time entry are quantified, discarded otherwise. However, the quantitation of molecules without retention time consumes more time.
Processors for quantitation: The amount of processors that shall be used for the quantitation. LDA detects the amount of processors available, automatically. The default value is always n-1, and the software does not allow for more than n processors.
Start Quantitation: This button starts the quantitation.
After the quantitation has been started, first Thermo RAW format and the Waters Mass Lynx raw directories are translated in mzXML (if the quantitation is based on the raw file formats). Second, mzXML files are translated in the internal chrom file format. If the raw file is Thermo RAW or Waters Mass Lynx and not mzXML, the intermediate mzXML is deleted automatically after conversion to chrom format. Then, on the chrom files, the quantitation is started. The results of the quantitation will be stored in an Excel file, whereby the name of the results file will be $path_of_the_chrom_file_without_suffix$_$quant_file_name$ (e.g. raw file: F:\lipidomics\20100210\20100126_TAG-34.chrom quant file: F:\lipidomics\TG_NH4_ACN.xls → result file: F:\lipidomics\20100210\20100126_TAG-34_TG_NH4_ACN.xls).