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Topic: Internal Standard Question and "Find" option in MS DIAL v4.16 (Read 87 times) previous topic - next topic

Internal Standard Question and "Find" option in MS DIAL v4.16

Hello,
I am having some difficulty understanding what input values to place when attempting to normalize data. Using a SPLASH mixture of known concentration, when I select to normalize the data there are default values in the "SPLASH mixture settings" window. Should I change the values according to how much was spiked into the sample? When inputting the values, do I need to take into account injection volume?
Also, when attempting to use the "Find" button, several, if any of the standards do not appear in the list or are labeled "w/o MS2". However, if I were to manually look for them in data acquisition software I am able to locate the precursor and MS2 spectra of the standards of interest.

Thanks.

Re: Internal Standard Question and "Find" option in MS DIAL v4.16

Reply #1
Hi members,

I would like to write the explanation of SPLASH lipidomics based quantification in MS-DIAL lipidomics project.
Actually, MS-DIAL supports three types of SPLASH mixture as the internal standards right now.
SPLASH LIPIDOMIX: https://avantilipids.com/product/330707
SPLASH II LIPIDOMIX: https://avantilipids.com/product/330709
EquiSPLASH: https://avantilipids.com/product/330731

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

C(lipids) = C(IS) * h(lipid)/h(IS) = 7.76 * 10,000/20,000 = 3.88 µM (in plasma)

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.

Thanks

Hiroshi