The primary function of LipidomeDB DCE is to search for m/z and signal for entered target lipid species in integrated (centroided) mass spectra uploaded by the user, to isotopically deconvolute the signals, and to quantify the signals in comparison to internal standard signals and amounts, in order to calculate the amount of each target lipid species in the samples. LipidomeDB DCE has three phases, shown in , data collection, data analysis, and result output and collection.
Scheme indicating the workflow of the system and relationships of the three phases of LipidomeDB DCE: data collection, data analysis, and result output and collection
To use LipidomeDB DCE, a login ID can be acquired by following instructions on the home page. A “tutorial” (i.e., documentation) accessible from the home page includes complete instructions for the use of LipidomeDB DCE. The instructions include brief directions for performing the lipidomic experiments, including suggestions for sample preparation, internal standards and their addition, acquisition of the mass spectral data and formatting of the mass spectral data for input into LipidomeDB DCE. The intent of this part of the tutorial is to simplify and describe the process of performing lipidomic analyses. Also available, on the home page and by following links in the tutorial, are example input data files, acquired on mouse intestinal lipid extracts, and the output file that should be obtained when the data are processed according to the instructions, allowing users to test the interface function and verify correct usage. Step-by-step directions for using LipidomeDB DCE (as shown in ) with screenshots are provided in the tutorial. Also in the tutorial are sections discussing normalization to tissue metrics and practical considerations about the limitations of the quantitative capabilities of direct-infusion electrospray ionization QqQ MS-based analysis.
Categories of step-by-step directions in the LipidomeDB DCE tutorial
The input to LipidomeDB consists of lists of target lipids, i.e., lipid species being analyzed, lists of internal standards utilized, internal standard amounts, and the mass and signal lists from precursor or neutral loss scan(s) (). Either pre-formulated lipid species target lists, user-specified lipid target lists or a combination of the two types of lists can be used. If the user composes his own sheets, they can be saved for his later use. The pre-formulated target lists for “plants” and “animals” are based on lipid species detected by the Kansas Lipidomics Research Center's analytical laboratory by electrospray ionization QqQ MS in a number of higher plant and mammalian species. is a screenshot of the interface for indicating the target lipids and the corresponding internal standards. Similar pages are used for each group of lipids to be analyzed (i.e., for each precursor or neutral loss scan). The pre-formulated lists provide a suitable and quick starting point for many users. Amounts of internal standards are entered, and mass spectral data are uploaded in Excel files. The interface for uploading the mass spectral data is shown in . LipidomeDB DCE searches the m/z of mass spectral data for the m/z values of lipids and internal standards in the target lists.
Screenshot of target compound input sheet. The screenshot shows the pre-formulated list for “Animal LPC”
Screenshot of spectral files upload sheet
Data Calculation and Output
Data calculation is described briefly in the “Materials and Methods” section, and in more detail in the tutorial. The output of LipidomeDB DCE can be viewed on (1) a sheet showing calculations for one target list for one sample, (2) as a summary showing calculated data for one target compound set from all samples or (3) as a summary of data for all targeted compound sets from all samples (). The sheet showing calculations for one target list for one sample (i.e., data from one spectrum) allows the user to confirm detection of the internal standards, to view the corrections for isotopic variants (isotopic deconvolution results) and to view the results of the quantification. The summary sheets provide the data in several convenient forms. All sheets can be viewed on the web or exported in Excel format.
Screenshot of summary. A summary of data for all targeted compound sets from all samples can be exported as an Excel file
LipidomeDB DCE is a well-documented web application for analysis of direct infusion electrospray ionization QqQ mass spectral data, with the capability to analyze multiple scans from up to 35 samples simultaneously. The pre-formulated lists of plant and animal polar lipids provide a quick start for lipidomic data analysis that should provide for ease in performing lipidomics analyses. In the long term, the web-accessible lipidomics data calculation system and database can be developed for storage and retrieval of lipidomics data in the context of experimental metadata.