6. Data analysis

macroMS provides limited data analysis reporting ion counts at defined m/z values for peaks within mass spectra of short mass range (m/z 1000). There are three input files that are uploaded through the web browser.
1. MALDI ToF MS spectra from ultrafleXtreme™
This data analysis function is for finding analytes with specific compounds. Therefore, data analysis pipeline forces m/z range limit of 1000 for all spectra. For example, if mass spectra ranges from m/z 200 to m/z 1750, the data analysis will not proceed because the spectrum range is 1550.
The server takes a single zipped file containing all of the output files. Data should be zipped from a single folder containing the data files, as shown in the following. Note: do not manually edit any part of the data file before submitting to data analysis. The max file size is 3.5 GB.
2. Image file
The exact image file used for generating the geometry file listing the targets.
3. Text file for m/z ranges of interest
Text file listing m/z value ranges for peaks of interest (up to 10) is needed. The file should be tab-delimited list of peak m/z ranges. The analysis rejects a text file containing a m/z range that is deliminated by whitespace (The space key). To have tab-delimited entries, the first m/z value is typed, then press the Tab key, type the second m/z value, then press the Enter key. Alternatively, two columns of m/z values typed in Excel spreadsheet can be cut+pasted to the text file, resulting in tab-deliminated entries.
Peak ranges are manually obtained from flexAnalysis like below.
250, 250.3 are the values for the above peak range
Greater the range is, more robust against variability of peak m/z value (due to sample height differences, etc), but increased chance to include a different peak.
MacroMS provides peak alignment functionality. Peak alignment assumes there is no contaminating peak within +/- m/z 0.5 from the exact mass for the calibrant, and the peak for internal calibration must appear in all of the mass spectra submitted. The mass spectra within +/- m/z 0.5 from the internal calibrant is smoothed using uniform_filter1d function of Scipy and the m/z value for the max intensity of the mass spectrum slice is taken as the tip of the peak. This value is compared to the exact value for calibration.
The exact mass, not molecular mass, for up to two internal calibrants are written within the text file like below. To indicate m/z ranges for peaks of interest, exact mass for the compound of interest and 1/2 of the width of the peaks are listed. 1/2 of the width of the peak is obtained from manual inspection of ~10 peaks in flexAnalysis. Again, the file should be tab deliminated.
Analysis
After uploading data and the email address, email will be sent to the address after completion of data analysis. This can take a few minutes to several hours depending on the size of the MALDI data file.
Output data: Excel file listing ion counts
The email contains a link to the excel file listing the analysis results which are summed ion count over the m/z values provided in the text file. Also, the file lists pixel coordinate and the size of the targets. Using Excel functions, data analysis can be performed by the user to find analytes of interest.
Output data: Data visualization tool
The second link included in the email is the link to the data visualization page. Once samples of interest are found by performing excel data analysis, the Blob ID or Group ID for the samples of interest can be cut & pasted into the input box for the visualization page. Single sample ID or single group ID can be selected from the drop-down selection list, which will trigger the viewer to zoom into the sample(s). The image of the samples is saved by taking the screenshot by user and saving to a software such as Powerpoint file.
To plot the MALDI-MS spectrum for samples of interest, select samples by using the drop-down menus or by the text input box. Set the m/z ranges to plot using the provided checkboxes, and click the plot button. To plot multiple samples, insert multiple samples in the text input box. The grayed out region is where the ion counts are summed for generating the values listed in the excel file.
Data plot mode
There can be external data analysis software that can analyze data generated from target list created by macroMS. Also, the macroMS data plotting page expires 3 days after data submission. To locate the analytes in the image and plot the associated data for the analytes for these cases, this data visualization functionality is provided.
Tab deliminated list of targets and its associated values can be copied from the Excel data file and put into the input box. The file name is underscore deliminated such as: 0_0_1829.02_1906.76_2_1.0. The file names are names of the data files generated from the macroMS target list. Submitting just the file name creates green colored boxes on the target. Submitting the file name and associated value result in blue-red heatmap.