Hi, I have actually realized this issue recently. The problem is that the algorithms to retrieve (A) the representative spectrum from samples and (B) the quant mass from samples are different and independently performed. Therefore, I should modify the program to (for example) retrieve the quant mass candidate from the masses included in the representative spectrum. I will fix the program till the next update. Hiroshi
Please see below for the description of the current MS-DIAL program.
(1) the user-defined mass error (e.g. 0.01 Da for MS1 tolerance) is converted to "PPM" unit considering 500 Da as the target mass. (2) if the mass is greater than 500, the mass tolerance is newly calculated as follows (see below).
User defined tolerance 0.01 Da Converted to ppm: 20@m/z 500
(3) the converted mass tolerance is used for (1) the MS/MS spectral assignment to precursor ion and for (2) the m/z filter in searching spectral libraries for compound annotation. *Note that the mass tolerance for <500 Da is not converted accordingly in the current program. It means that the mass tolearnce is set to 0.01 Da for <500 Da metabolites in spectral searching.
In principle, ABF file converter retrieves the calibrated data. Therefore, the abf format file should contain the calibrated data if you reflected the calibration to your raw data in the DataAnalysis software of Bruker.
I will activate newly added lipid subclasses described in the BioRxiv reprint at the next update of MS-DIAL. I am now planning to post the new version of MS-DIAL on the beginning of March. Sorry for your inconvenience.
If you go to "data visualization"-> "normalization" -> "SPLASH", you will see the image like "image1.png".
* Importantly, the default values are based on our experimental protocol where 5 uL of EquiSPLASH original mixture is added for 20 uL plasma. But there are several output options in MS-DIAL like pmol/10^6 cells, or pmol/mg tissue.
What you have to set is to set the concentration of lipids as pmol/mg tissue or something like that. Below is the example for plasma lipidomics choosing "pmol/uL plasma" as the unit output. What you will do is: 1. Calculate the absolute amount of IS in pmol that is in the extraction solvent volume 2. Calculate the concentration of IS in pmol/µL plasma based on the volume of plasma used for the extraction 3. For single point calibration, MS-DIAL use the ratio of peak heights of the lipids and IS and multiply by the concentration of IS in plasma
Below is an example calculation methodology for concentration which was given from Tomas Cajka (Lab of Metabolomics at Institute of Physiology CAS):
• TG-IS: 194 pmol abs. amount (in 165 µL MeOH used during the extraction) • Plasma volume for the extraction: 25 µL • 194 pmol/25 µL = 7.76 pmol/µL plasma = nmol/mL = µmol/L = µM • h(lipid): 10,000 • h(IS): 20,000
Note 1: Users have to set their own values for concentrations. Again, the default values are based on our experimental protocol where 5 uL of EquiSPLASH's original mixture is added for 20 uL plasma. Therefore, if you added 5 uL of EquiSPLASH for 10 mg liver tissue of mouse, all values should be changed to 1/2 x the default value.
Note 2: To efficiently use the "Find" button, you need a trick. To "find" the internal standards in your data, the annotated name must be equal to the names shown in MS-DIAL SPLASH lipidomics normalization window. Therefore, please use the attached template files which can be imported as the text library in the identification tab of analysis parameter setting. (Please correctly set the retention times for those lipid standards for your own experimental condition.) Once you annotate the detected peaks by the lipid names included in the template text library, you will be able to use the find button effectively. Otherwise, please manually set the alignment IDs correspond to the peaks of internal standards.
in GNPS export option, to exclude the blank sample features from the data matrix, you should not tick the option of "Keep removable features and assign the tag" of Alignment tab. By this, blank features will be excluded from the result of peak alignment. For CAMERA-like function, MS-DIAL has the following four annotation procedures as the post curation of peak detection and alignment result to provide the peak characters. See also: https://www.nature.com/articles/s41592-019-0358-2 1. Chromatogram-based annotation: peaks having similar chromatographic peak shapes in the same RT area are grouped. 2. Adduct annotation: adduct ions from a metabolite are grouped. 3. MS/MS based annotation: The precursor ion observed as a product ion of the higher m/z's MS/MS spectrum is tagged as a tentative candidate of ion source fragment ion of a precursor ion. 4. Alignment-based annotation: the correlation of ion abundances of two alignment spots was calculated for all precursor ion pairs found in ±0.02 min. The spots were assigned the term ‘highly correlated ions’ when the correlation coefficient was greater than 0.9.
Such annotations are also exported as the edge files of GNPS export, and they can be used in GNPS IINxFBMN platform, which will be supported in very near future. Meanwhile, could you please check these edge files to reduce the nodes which should have same MS/MS features? Also, well, MS-DIAL MS2Dec cannot provide a good MS/MS spectral separation (deconvolution) if the peak tops are completely same. In such case, CorrDec function should be useful to provide the decovoluted spectra using the ion abundance correlations across biological samples. Ipputa Tada who is the responsible person will put more information on this chat.
New features of MS-DIAL: 1. Modified IBF converter program to accurately calculate the decimal of ion mobility data's m/z values for accumulated MS1 spectra. 2. The normalization functions are now fully supported in ion mobility data processing. 3. Fixed the theoretical spectra of [M+Na]+ ion form of LPC and PC. 4. Fragment rule based lipid annotations were improved for several lipid subclasses especially in [M+Na]+ form. 5. Improved and fixed the programs from the feedback in MS-DIAL software forum.
I could reproduce the issue, and I fixed the program. (next week, I will upload the new version of MS-DIAL.) Meanwhile, please do not tick "Use reference spectrum to make MRMPROBS library" if your spectrum is "unknown". The program will try to use the reference (library) spectrum to generate MRMPROBS library file if it is ticked. If you do not tick it, MS-DIAL will use the experimental spectrum to generate the MRMPROBS library. Ane no error is occurred in the current version.
Thanks, this was the invalid file name characters issue due to "|" in the lipid metabolite name to be used as the file name of png and emf. I fixed it, and I will upload the latest version in next week actually. Thanks,
please follow the direction of Biswa. Also, the file of "GMD_20111121_VAR5_ALK_MSL.txt" in GOLM database website should be good to start to merge the library with the existing spectral records on my website.
as long as I understand your data, please follow the below setting. 1. Capture.PNG shows the example startup project window for your data. Note: SWATH-MS or conventional all-ions method is fine for your data actually. (although in fact you may use both methods) All-ions with multiple-CEs setting is actually made for the method incorporating three or more collision energies for all-ions like 0 SCAN 100 1500 MS1 6 1 1 ALL 100 1500 MS2 20 1 2 ALL 100 1500 MS2 50 1 3 ALL 100 1500 MS2 70 1 However, your data is obtained by two CEs setting. So the experimental file should contain the following information (as attached as experimental_file.txt).
ID MS type Start mz End mz 0 SCAN 100 1500 1 MSE 100 1500
The other columns like Name and CollisionEnergy are not used in "SWATH-MS or conventional all-ions" method option.
2. Use abf file format for your analysis actually. (I personally have never checked the process by mzML...) 3. I recommend to optimize (A) minimum peak height of peak detection tab (B) MS/MS abundance cut off of MS2Dec tab for your analysis to rapidly process your data. Especially check the baseline of noise signals. According to my experience for synapt g2 mse, the baseline of noise signal is around 10^5 - 10^6, and therefore, I used around 10^5 value for (A) and (B) parameters. If you use the default setting (1000 and zero for A and B), the processing time requires too much hours because it should also handle the noisy spectra.