Manual for MFSearcher toolversion 1.6.0
August 28, 2019
License
MFSearcher GUI tool is available free for charge for academic purpose. The compounds
registered in the following databases can be searched by mass values using this tool.
Please preserve the license information of each databases when using the search resultsof this tool.
KEGG

http://www.genome.jp/kegg/

KNApSAcK

http://kanaya.naist.jp/KNApSAcK/

FlavonoidViewer

http://metabolomics.jp/wiki/Category:FL

HMDB

http://www.hmdb.ca/

LipidMAPS

http://www.lipidmaps.org/

UNPD

http://pkuxxj.pku.edu.cn/UNPD/

PubChem

http://pubchem.ncbi.nlm.nih.gov/

Requirement sPC (Windows is recommended) in which Java runtime environment (1.7 or later) isinstalled and connected to the Internet is required.
The software accesses to the Internet in the following operations:- When executing the search, the tool accesses to the MFSearcher web service(http://webs2.kazusa.or.jp/mfsearcher/) in the background process.
- When browsing the searched compound information, the tool accesses to the original
databases by the Internet browsers.
If proxy server is used to connect to the Internet, please set the proxy serverinformation according to the section “Proxy Server Settings”.
Please refer to the URL below for installation of Java.
https://www.java.com/ja/download/help/download_options.xml
1Run/Stop the tool
- Run as a GUI tool
1) Decompress the downloaded .zip file. The following files and folders are appeared.

2) Double click the "MFSearcherRun.bat" file to run the tool. The main window ofMFSearcher GUI is displayed.

* A console window shown below is appeared too. Don't close this window when
MFSearcher program is running. If the window is closed by clicking "X" button on
top-right of the window, the MFSearcher program stops too.

3) To stop the tool, click the "X" button on top-right of the main window.

2- Run as a command line tool
Youcanseeahelpofthecommandline

toolbydouble

clicking

the“MFSearcher_CommandLine.bat” file in the extracted file set (see above). The commandline tool is useful for batch processing for a large number of mass values (and theirestimated adducts). The results are obtained as text files.
Details of the command line tool are described in the “Command line mode” section.

Basic Use
1) Enter search conditions.
adduct: Select a type of adduct.
mass: Enter the mass value
margin: Enter the mass tolerance, and select the units, ppm or m/z (Da).

2) Check the databases for search.

Following

databases

canbeused.

See

also

MFSearcher

website(http://webs2.kazusa.or.jp/mfsearcher) for the details.
EX-HR2A database of computationally calculated elemental compositions3

of possible molecules with correct valences.
Pep1000

A database of computationally calculated linear polypeptides
whose molecular weight are up to 1000.

KNApSAcK

A database of natural products.
http://kanaya.naist.jp/KNApSAcK/

KEGG

Compound database in the KEGG pathway.
http://www.genome.jp/kegg/

PubChemA huge compound database maintained by NCBI.
https://pubchem.ncbi.nlm.nih.gov/

HMDB

A database of compounds detected and predicted in human body.
http://www.hmdb.ca/

FlavonoidViewer

A database of flavonoids. Flavonoid is a group of secondary
metabolites primarily biosynthesized in plants.
http://metabolomics.jp/wiki/Category:FL

LipidMAPS

A database of lipids, polyphenols and so on.
http://www.lipidmaps.org/

1) Execute searching by clicking the "Search" button. The results are listed in the table.

Click one of the results to see the name of the compound. The name is displayed in thetext field at the bottom of the main window.
Click the "Link" button to see the information of the compound. Web browser will open
and the original page of the selected compound in the database can be seen.

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UC2 Search
UC2 database compiles the information of the same compounds in multiple databases
into one result. UC2 has following features too:- Charged compounds in the original databases, such as cyanidines ([M]+), are searchedwith correct charge states and mass values.
- Compounds registered as multiple components in the original databases, such as salts,
are searched as a single component (the component of the largest molecular weight isused).
Therefore, UC2 search excludes the miss hits to the charged compound and salts, andreduce the candidates.
* The identity of the compound structure is judged by the connectivity of the atoms
using the first block (14 letters) of InChIKey, and the stereochemistry is notdistinguished. For example, D-type and L-type of an amino acid is compiled in a singleresult.
1) Set the adduct, mass and margin, same as those in the normal search.

2) Check the "UC2" radio button to enter the UC2 mode.

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In UC2 mode, the databases, EX-HR2 and Pep100, cannot be selected and searched,because these databases does not contain chemical structural information. The UNPD
database can be selected in UC2 search.
UNPD

A large compound database of natural product
http://pkuxxj.pku.edu.cn/UNPD/

3) Click "Search" button to execute the search. The results are listed in the table.

In UC2 mode, the result table is separated into two tables. The left table showed the listof the same connectivity where several compounds are compiled. Click one of the list,
then the database names and the IDs of the compiled compounds are displayed in theright table.
Select a compound in the right table, and click "Link" button. The details of thecompound information can be read in the Internet Browser.
* Records in the UNPD are not linked because of the specification of the originalwebsite.
The compound name displayed in the left table is a representative of those that has theshortest text length among the compiled compounds.

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The column “Char.” in the right table shows the features of the compounds in theoriginal database. Each letter in the column shows followings:
NumberThe charge of compound.

fThe compound was registered as multiple components, such as a salt.

rThe compound was a radical.

When the query is recognized as apparent molecular ion, the information of themolecular ion is displayed in the Formula, Adduct and FW (molecule) column. For
example, when a mass is searched as [M+H] + and all the results are records with +1
charge, the information of [M]+ will be displayed.

When all the results have opposite charge to the queried adduct, a candidate adduct ionwith “?” will be displayed in the Adduct column.

-1+1Advanced Settings

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Click the "menu" button to show the menu.

Following items are included in the menu.
View

Show/Hide Status Bar

This menu item switches On/Off of the statusbar at the bottom of the main window.

Show DB Info.

A window that informs the record numbers
and updated dates of the databases will open.
* The databases listed here is only for thenormal search. The data used in UC2 search
are not the same one of them and are notdisplayed here.

Setting

Hide Menu

This menu item hide the menu.

Column Settings

Users can change the columns in the resulttable.

Filter Settings

A setting window for filtering the results byatom numbers etc., will open.

Filter Settings
Open a setting window by selecting "Filter Settings" in the "Setting" menu.

81) Filtering by atom numbers
Users can filter the results by number of atoms C, N, S, and P in the chemical formula.
Check the check boxes adjacent to the atom labels to enable the filtering. The minimumand maximum number of the atoms are set by the text fields at left and right of theatom label. An optional atom label can be set in the text field under "P".
Check the "Constraint only for EX-HR2 DB" to limit the filter settings only to theEX-HR2 database.
2) Filtering by 13C/12C ratio
Users can filter the results by formula that match to the ratio of the intensities of 13C1
stable isotope peak and to 12C monoisotopic peak.

Enter the 13C/12C ratio (obtained by experimental measurement etc.) in the text field atthe left.
Enter the tolerance of the error in % in the text field at the right.
Check the check box "Show only 13C in" to enable the filtering.
3) Limit the FlavonoidViewer results to aglycones
Check the " Show only aglycon" at the "FlavonoidViewer Option" to limit the results
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from FlavonoidViewer to aglycones.

Command Line Mode
Command line mode is useful for batch processing for a large number of mass values(and their estimated adduct ions) for formula prediction and compound databasesearching. Results are obtained as text files.
1) Preparation of an input file
A list of mass values is prepared as a text file. Following three formats are acceptable.
You can see examples in the sample_file folder.
a) A list of mass values (input_text_plain.txt)
Mass values are written in each line. The mass values are directly used for calculation.
b) A list of pairs of mass value and adduct (input_text.txt)
Pairs of mass value and estimated adduct ion are separated by tab and written in eachline. The labels of the adduct ions should be defined in “adduct.ini” file. See “AdductSettings” section for details.
c) MassChroViewer CheckFile (input_mcv_checkFile.txt)
A file used in the MassChroViewer tool. The file is generated by MassChroViewer andPowerGetBatch.
MassChroViewer: http://www.kazusa.or.jp/komics/software/MassChroViewer
PowerGetBatch: http://www.kazusa.or.jp/komics/software/PowerGetBatch

102) Run command prompt tool (terminal tool) of the OS.
3) Using “cd” command, move to the directory where you have extracted the fileset ofthe tool. You can see “MFSearcher.jar” file there.
4) Command line help is available by executing following command.
java -jar MFSearcher.jar -h5) Similarly, you can execute MFSearcher tool in command line mode by defining theinput file, output directory, compound databases for search, mass tolerance for formulaprediction and database search as options.
Ex) A case using a list of pairs of mass value and adduct ion (input_text.txt)
java-jarMFSearcher.jar-i

sample_file/input_text.txt-odoutdir_plain-db

KG,KN,FL,HM,LM -fppms 1,3,5 -dppms 3,5,15
Multiple values for mass tolerances can be set by separating the values by comma. In
this case, formula prediction and/or database search are attempted using smaller
tolerances and continued till any result obtained. The UC2 database is used fordatabase search.
6) Result files are obtained at the directory defined by “-od” option.
ResFormula_[Input File Name]: Full results of formula prediction.
ResDatabase_[Input File Name]: Full results of database search.
ResSummary_[Input File Name]: Summary of the two files above.

Adduct Settings
See the "conf" folder in the file sets of MFSearcher program.

11A file named "adduct.ini" is included in the conf folder. By editing the adduct.ini file, theinformation of adducts used in the MFSearcher program can be changed.
The adduct.ini file is written in a tab delimited text format. Use a proper software suchas a text editor (note pad) to edit the file.
An example of the file structure is as below.
1) Caption2) Number ofmolecule3) Formulaadded124) Formulasubtracted5) Optional
mass value

6) Charge

7) Not used1) An arbitral name can be set. Avoid to enter the same name in multiple rows.
2) In the case of [2M+H]+, 2 should be entered.
5) If the mass difference is difficult to described by formula, enter a numerical valuehere.
7) This column is not used.
Save the file and restart the MFSearcher tool to reflect the editing.

Proxy Server Settings
See the "conf" folder in the file sets of MFSearcher program.

Find a file named “system.ini” in the “conf” folder. Open the “system.ini” file by a text
editor (such as Note Pad). A default setting is shown below.

Edit the text in the right side of “=” as below.
network.proxy.enabledtrue

network.proxy.serverName or IP address of the proxy server

network.proxy.port

Port number of the proxy server

Example)Overwrite the “system.ini” file, and then restart the MFSearcher.
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Publications
Sakurai N, Narise T, Sim JS, Lee CM, Ikeda C, Akimoto N and Kanaya S (2018) UC2
search: Using unique connectivity of uncharged compounds for metabolite annotation
by database searching in mass spectrometry-based metabolomics. Bioinformatics 34:
698-700[PMID: 29040459]
Sakurai N, Ara T, Kanaya S, Nakamura Y, Iijima Y, Enomoto M, Motegi T, Aoki K,Suzuki H and Shibata D (2012) Application of a relational database system forhigh-throughput prediction of elemental compositions from accurate mass values.
Bioinformatics 29 (2): 290-291[PMID: 23162084]

Contact Information
National Institute of Genetics, Mishima, Japan
Nozomu SAKURAI Ph.D.
E-mail: sakurai AT nig.ac.jp (replace AT with @)The earlier version of this tool was developed by NS at Kazusa DNA Research Institute,
Kisarazu, Japan.

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