Background : Once you have processed LCMS data files with the MS-DIAL software, you have a list of several thousand unknown peaks with MS/MS spectra. A candidate list of probable compounds can be generated using the mass to charge ratio and the retention time for those peaks. The next task is to match the predicted spectra for those candidate compounds with the experimental spectra of peaks. A good matching score will provide a better confidence in the annotation. It is a good way to get rid of many false positive hits. However,  the compound’s pure standard needs to be purchased and RT and the MS/MS need to be validated for yielding a compound that can be included in the metabolomics data matrix for statistics. Below example shows how to use the MS-Finder software to match the predicted and experimental spectra and to perform sub-structure annotation for product ions.

Example spectra of Tryptophan was used for this demo.  

Step 1. Download the MSFinder software

http://prime.psc.riken.jp/Metabolomics_Software/MS-FINDER/

http://prime.psc.riken.jp/Metabolomics_Software/MS-FINDER/index2.html

Step 2. Prepare the MSP file

Input : save below spectra  as ‘tryptophan.msp’ in a new folder.

NAME: L-Tryptophan

PRECURSORMZ: 205.0972

PRECURSORTYPE: [M+H]+

INSTRUMENTTYPE:

INSTRUMENT:

Authors:

License:

FORMULA: C11H12N2O2

ONTOLOGY:

SMILES: C1=CC=C2C(=C1)C(=CN2)CC(C(=O)O)N

INCHIKEY:

INCHI:

IONMODE: Positive

COLLISIONENERGY: 40

SPECTRUMTYPE: Centroid

METABOLITENAME:

SCANNUMBER: 0

RETENTIONTIME: 0

RETENTIONINDEX: 0

INTENSITY: 0

#Specific field for labeled experiment

Comment:

MSTYPE: MS1

Num Peaks: 0

MSTYPE: MS2

Num Peaks: 41

50.6945        0.38

52.1479        0.44

52.1579        0.41

52.2037        0.44

52.2424        0.42

54.0548        0.92

55.9309        0.47

62.8003        0.66

73.8515        0.51

74.0243        10.24

74.1591        0.55

77.6211        0.48

77.7631        0.49

86.0606        0.62

87.0446        6.92

88.6855        0.49

91.0546        1.04

115.0543        0.93

117.0572        1.03

117.0701        1.03

118.0652        25.16

130.0651        8.11

132.0808        38.58

140.1044        0.49

142.0652        6.6

143.0723        5.35

144.0443        6.65

144.0807        107.06

146.06        617.11

146.1177        0.62

147.063        0.54

149.0233        2.9

159.0916        82.5

160.0756        8.47

170.0599        30.77

187.9819        0.64

188.0706        999

188.1555        0.74

189.0733        0.58

205.0766        1.2

205.097        9.13

Note : MSFinder can process more than one spectra in batch mode.

Step 3. Start the MSFinder software :

It will take couple of seconds to load the libraries and structure database.

Select the folder containing the MSP file –

MSFinder will import the MSP file. It should look like this –

Step 4. Annotate product ions.

Click on analysis → Peak assignment (batch job)

Step 5. Load the annotation. Import again the folder containing the MSP file.

Bottom right plot shows the fragments matched to the experimental spectra. Hovering mouse pointer on fragments shows the substructure annotations.

MSFinder creates a .sfd file inside a sub-directory.

Inside this folder, C11H12N2O2.sfd file has the SMILES annotations for product ions. This file can be imported into a database.