ms-finder tutorial - prime: platform for riken...
TRANSCRIPT
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MS-FINDER tutorial
Last edited in November 9, 2016
Abstract
The purpose of metabolomics is to perform the ‘comprehensive’ analysis for small biomolecules of
living organisms. Gas chromatography coupled with electron ionization mass spectrometer (GC/MS)
and liquid chromatography coupled with electrospray ionization- (ESI-) tandem mass spectrometer
(LC/MS/MS) are the preferred tools for untargeted metabolomics. Currently, the main bottleneck of
GC/MS- and LC/MS/MS based untargeted analysis is compound identification due to the limitation of
EI-MS and MS/MS records of authentic standard.
MS-FINDER was launched as a universal program for compound ‘annotation’ that supports
EI-MS (GC/MS) and MS/MS spectral mining. First, MS-FINDER aims to provide solutions for 1)
formula predictions, 2) fragment annotations, and 3) structure elucidations by means of unknown
spectra. In addition, the program can annotate your unknowns by the public spectral databases such as
MassBank, LipidBlast, and GNPS. MS-FINDER has been developed as the collaborative work
between Prof. Masanori Arita team (RIKEN, Reifycs Inc.) and Prof. Oliver Fiehn team (UC Davis)
supported by the JST/NSF SICORP ‘Metabolomics for the low carbon society’ project.
Hiroshi Tsugawa
RIKEN Center for Sustainable Resource Science
MS-FINDER screenshot
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Table of contents
Software environments .............................................................................................................................. 3
Required programs ..................................................................................................................................... 4
Acceptable ASCII formats .......................................................................................................................... 5
MSP format for MS/MS .......................................................................................................................... 6
MSP format for EI-MS ............................................................................................................................ 7
MAT format ............................................................................................................................................. 8
Adduct ion format: [M+Na]+, [M+2H]2+, [M-2H2O+H]+, [2M+FA-H]-, etc. .................................... 10
User defined structure database format ............................................................................................. 11
Import queries ........................................................................................................................................... 12
A. From a folder which includes MSP or MAT format files ............................................................... 12
B. From the graphical user interface of the MS-FINDER program .................................................. 13
C. From the MS-DIAL program ........................................................................................................... 14
Parameter setting ..................................................................................................................................... 15
Method tab ............................................................................................................................................. 15
Mass spectrum tab ................................................................................................................................ 16
Formula finder tab ................................................................................................................................ 17
Structure finder tab .............................................................................................................................. 18
Compound annotation by in silico fragmenter ....................................................................................... 20
Compound annotation by searching spectral databases ........................................................................ 22
Compound annotation (batch analysis) ................................................................................................... 23
Peak assignment (single) ......................................................................................................................... 24
Peak assignment (batch job) .................................................................................................................... 25
Mouse function .......................................................................................................................................... 26
Export ........................................................................................................................................................ 27
Help ............................................................................................................................................................ 28
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Software environments
Windows OS (.NET Framework 4.0 or later): Windows 7 or later
RAM: 8.0 GB or more
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Required programs
MS-FINDER
Download link: http://prime.psc.riken.jp/Metabolomics_Software/MS-FINDER/index.html
MS-FINDER can be used as the local software program in Windows PC. The program can
import ASCII format files including MSP (EI-MS and MS/MS) or improved MSP (both MS and
MS/MS, the file extension must be MAT.). In addition, the users can directly make the query in
the MS-FINDER graphical user interface. Moreover, this program can be called from the MS-
DIAL program which is downloadable at http://prime.psc.riken.jp/Metabolomics_Software/MS-
DIAL/index.html.
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Acceptable ASCII formats
This program accepts two file extensions, i.e. MSP or MAT formatted by the following explanations.
Unknown queries should be separately stored in the ASCII file: the MSP or MAP file ‘CANNOT’
store multi compound records in the single file.
The format of MSP basically follows the NIST MS search manual.
Link: http://www.nist.gov/srd/upload/NIST1a11Ver2-0Man.pdf
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MSP format for MS/MS
Required fields
NAME:
PRECURSORMZ:
PRECURSORTYPE:
IONMODE: (Positive or Negative)
Num Peaks:
m/z intensity pair (tab, comma, space can be used as the delimiter.)
Mass spectrum is supposed to be imported as MS/MS.
If you want to perform the MS/MS peak annotation with the known structure, prepare two fields
including FORMULA and SMILES. Please note that the formula and SMILES of the neutralized
structure should be prepared.
MSP example
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MSP format for EI-MS
Required fields
NAME:
IONMODE: (Positive or Negative)
Num Peaks:
m/z intensity pair (tab, comma, space can be used as the delimiter.)
The fields are the minimum requirement for searching spectral databases. In the case that you want
to perform formula predictions and structure elucidations in EI-MS data, two files
‘PRECURSORMZ:’ and ‘PRECURSORTYPE:’ must be required.
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MAT format
The MAT format was defined as the improved version of MSP in the MS-FIDNER program to store
both MS1 and MS/MS spectra in the same file. The survey scan MS data should be required to
calculate ‘isotopic ion score’ for formula predictions. Importantly, for EI-MS spectra, put your
spectra into both MS1- and MS2 fields for the calculation of isotopic ratio and fragment ion
similarities, respectively.
Required fields
NAME:
PRECURSORMZ:
PRECURSORTYPE:
IONMODE:
MSTYPE:
Num Peaks:
m/z intensity pair (tab, comma, space can be used as the delimiter.)
Three fields including MSTYPE, Num Peaks, and m/z intensity pair should be SERIALLY
stored.
If you type ‘MSTYPE: MS1’, the spectrum written from next field should be recognized as the
survey scan MS (MS1). If you type ‘MSTYPE: MS2’, next spectrum should be recognized as
the MS/MS spectrum.
EI-MS spectra must be stored in both MS1 and MS2, which is the requirement of MS-FINDER.
Both field (MSTYPE: MS1 and MSTYPE: MS2) is not necessary for this program, i.e. the users
can import the ASCII file as only MS1 spectrum or as only MS/MS spectrum record.
Users may prepare the MAT or MSP files without any spectrum record. In such case, the formula
prediction will be performed by means of mass accuracy and database criteria.
If you want to perform the MS/MS peak annotation with the known structure, prepare two fields
including FORMULA and SMILES. The formula and SMILES of the neutralized structure
should be made.
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MAT example
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Adduct ion format: [M+Na]+, [M+2H]2+, [M-2H2O+H]+, [2M+FA-H]-, etc.
1. The parentheses ‘[’ and ‘]’ must be used to bracket the ion information.
2. The char + and - must be required after ']' and the number must be written before + or -.
3. When you want to define the organic formula like C6H12O5, you have to write it without any
replicate elements or parentheses. For example, the descriptions like [M+C2H5COOH-H]- or
[M+H+(CH3)3SiOH]+ are not accepted.
4. The beginning figure of organic formula like '2'H2O is recognized as the H2O × 2. Again, never
use 2(H2O) for that.
5. Sequential equations are acceptable: [2M+H-C6H12O5+Na]2+ (very apt.)
6. Radical ion can be described by ‘.(dot)’ after + or – like [M]+. And [M-CH3]+. as adduct format.
7. MS-FINDER accepts some abbreviations or common organic formulas for adduct types as
follows.
For Acetonitrile: ACN, CH3CN
For Methanol: CH3OH
For Isopropanol: IsoProp, C3H7OH
For Dimethyl sulfoxide: DMSO
For Formic acid: FA, HCOOH
For Acetic acid: Hac, CH3COOH
For Trifluoroacetic acid: TFA, CFCOOH
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User defined structure database format
MS-FINDER supports the structure elucidations from the candidates that users provide. The
following format file should be prepared as tab-delimited text file. The identifiers of InChIKey, short
InChIKey, and database ID are not required, but the values must be filled by some mimic values. The
files of exact mass, formula, and SMILES must be prepared.
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Import queries
There are two ways to import unknown queries.
A. From a folder which includes MSP or MAT format files
1. File -> import
2. Select a folder containing MSP or MAT files.
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B. From the graphical user interface of the MS-FINDER program
1. File -> Create a query
2. Fill in the form to make a query.
Required files
Folder path
File name
Precursor m/z
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C. From the MS-DIAL program
The MS-DIAL program which has been reported as software for data processing of LC/MS/MS can
call the MS-FINDER program directly. On the first time when you call the MS-FINDER program at
MS-DIAL, please select the file path of MS-FINDER via GUI.
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Parameter setting
Method tab
MS-FINDER provides two options for compound annotation: one is by spectral databases, and the
other is by formula- and structure finder programs using in silico fragmenter. You can simultaneously
check both ‘spectral database search’ and ‘formula prediction and structure elucidation by in silico
fragmenter’ options, and the result of spectral database search has priority for ranking structures.
The ‘internal experimental library’ is stored in ‘EIMS-DBs-vs*.egm’ and ‘MSMS-DBs-vs*.etm’
of Resources folder as NIST MSP format. If ‘Formula finder > TMS-MeOX derivative compound’ is
checked, EIMS database will be used; otherwise, MSMS database is used. The ‘in silico library for
lipids (LipidBlast)’ is stored in ‘MSDIAL-LipidDBs-vs*.lbm’ of Resources folder. Select the appropriate
solvent condition for searching your unknowns. The user-defined spectral database must be formatted
by NIST MSP.
If ‘Precursor oriented spectral search’ is checked, the structure candidates will be filtered out
by the precursor m/z of spectral records in combination with MS1 tolerance value; otherwise, all of
spectral records will be used. Uncheck this option if you want to search EI spectral databases.
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Mass spectrum tab
Mass tolerance (MS1): the mass tolerance to generate formula candidates.
Mass tolerance (MS/MS): the mass tolerance for matching experimental- and reference fragments.
Relative abundance cut off: The product ions more than this parameter on the basis of base peak ion
are utilized for the product ion matching.
*For EI-MS spectra, set the same tolerance into MS1 and MS2.
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Formula finder tab
Formula calculation setting: You can set the parameters for formula calculation.
LOWIS and SENIOR check: to generate formula candidates that match the valence rules of formula
elements. The valences of hetero atoms, i.e. N, O, S, and P are currently set to 3, 2, 6, and 5,
respectively.
Isotopic ratio tolerance: to calculate the isotopic score. The tolerance should be utilized as the sigma
value for the Gaussian scoring as described in the MS-FINDER paper.
Element ratio check: to generate formula candidates that satisfy every element ratios (ex. H/C ratio
should be between 0 and 3.33 for ‘Common range (99.7%)’ restriction. ) as described in the MS-
FINDER paper.
Element probability check: to generate formula candidates that satisfy the heuristic rules as described
in the Seven Golden Rules paper. For example, if a formula candidate contains the following element
counts, i.e. NOPS all > 1, the element counts of N, O, P, and S should be less than 9, 19, 3, and 2,
respectively.
Element selection: to generate formula candidates that just contain the elements selected by the
users. Check ‘TMS-MEOX derivative compound’ if you want to annotate EI-MS spectra.
Result cut off: formula candidates ranked by the MS-FINDER program will be reported within up to
this number.
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Structure finder tab
Here is the parameter setting for in silico fragmenter.
Tree depth: the limitation of in silico cleavages, i.e. if the user sets ‘2’, the MS-FINDER program
generates the fragments until product ions of a product ion.
The current MS-FINDER program can utilize the fragment ion library for EI-MS spectral mining
which is stored in ‘EiFragmentDB_vs*.eif’ (recommended to use).
Result cut off: structure candidates ranked by the MS-FINDER program will be reported within up to
this number.
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Data source
Local databases: currently, total 14 metabolome databases are prepared in the MS-FINDER program
which is stored in ‘ExistStructureDB_vs*.esd’. The local databases selected by users will be used to
retrieve the structure data. Please see ‘user defined database format’ section for searching your own
structure candidates.
MINEs (Metabolic In silico Network Expansions) and PubChem online settings:
If the user selects ‘Never use it’, the structure candidates will be picked up just from the local
databases.
If ‘only use when there is no query in the below DBs’ is selected, the structure data of MINE and
PubChem compound databases will be retrieved when no structures can be found in the local
databases.
If ‘always use it.’ is selected, the MS-FINDER program always retrieve the structure data from
MINE and PubChem databases in addition to local databases.
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Compound annotation by in silico fragmenter
The general workflow of MS-FINDER is described here. (Batch analysis is shown below)
1. The formula prediction is executed by double click at the title name of ‘File navigator’. The
detail of formula calculations is described in our paper. *The point of formula prediction is to
correctly select the precursor type (adduct ion) and to set parameters for picking up the correct
formulas.
A) The formula candidate checked in ‘Select’ column is supposed to be examined at structure
finder program.
B) The isotopic ions will be displayed by ‘I’ button at the upper mass spectrum window.
C) The result of formula assignment in product ions will be displayed by ‘P’ button at the
bottom spectrum window.
D) The annotation result of neutral losses will be displayed by ‘N’ button at bottom window.
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2. The structure finder will be executed by right click at the formula result table followed by
clicking ‘Search the structure’.
A) The formula candidate checked in ‘Select’ column is supposed to be examined at structure
finder program.
B) Total score of structure finder is the total of formula- and structure scores. Therefore, even if
a formula score is greater than the others, the other structure from another formula candidate
may become the top candidate in the structure finder program.
C) The MS-FINDER program integrates the structures having the same molecular skeleton by
its InChIKey (first 14 characters). Its representative structure will be determined by the
number of synonymous in PubChem repository.
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Compound annotation by searching spectral databases
It’s very simple. Double click the unknown record that you want to annotate. The below is the
examples for searching EI-MS spectral database and LipidBlast database.
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Compound annotation (batch analysis)
1. Analysis -> Compound annotation (batch job)
A) If you want to perform the batch job for both formula predictions and structure elucidations,
please check ‘Both processes. Also, add the number in ‘Top N hits’ textbox where the formula
candidates generated by the formula finder program are supposed to be searched.
B) If you want to perform the batch job for formula predictions, please just check ‘molecular
formula finder’.
C) If you want to perform the structure finder program, please just check ‘structure finder’. Here,
the formula candidates checked by the users are supposed to be examined. Also, if the formula
finder is not executed before this analysis, the queries will be passed.
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Peak assignment (single)
The MS-FINDER program can be used as the peak assignment tool to assign substructures in the
MS/MS spectrum from user-defined structure.
1. Analysis -> Peak assignment (single)
2. Select the query file that you want to analyze.
3. Add both formula and SMILES into the textboxes as the neutralized form.
4. The result will be generated as shown below.
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Peak assignment (batch job)
Analysis-> Peak assignment (batch job)
To use this program, please make sure that the record in MSP or MAT files should contains the
respective FORMULA and SMILES fields. Otherwise, the program will be passed for records not
having their fields. The SMILES or FORMULA records can be added in ‘File information’
textboxes of MS-FINDER GUI.
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Mouse function
A) Mouse right click (or hold) and move: zoom in and out
B) Mouse left click (or hold) and move: select and scroll
C) Mouse left double click: reset range and select files in the file navigator
D) Mouse wheel: zoom in and out
E) Right click: popup context menu
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Export
The result of formula and structure finders can be exported from this option. Currently, the top 10
candidates will be automatically exported. However, you can also check the details of result as the
ASCII file. It means that the MS-FINDER program is supposed to generate the FGT file containing
formula results in the same directory as the project folder. Also, the program generates the respective
folder containing the SFD file (per formula candidate) which stores the result of structure finder
program.
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Help
You can check the version of MS-FINDER.