RMassBank: The workflow by example



RMassBank is a two-part computational mass spectrometry workflow:

This vignette describes basic usage with the standard workflow. The package is flexible and allows for different advanced use cases. Examples of specialized applications of RMassBank are available at the RMassBank message board hosted by the Metabolomics-Forum: (http://www.metabolomics-forum.com/viewforum.php?f=29).

Installation and loading

The library is available from Bioconductor ((http://www.bioconductor.org)). In addition to the library itself, it is recommended to install the OpenBabel chemical toolkit, available from (http://www.openbabel.org) for various platforms (or via Linux package distribution systems).

The library is loaded as follows


The data used in the following example is available as a package RMassBankData, which must be installed separately and is loaded using


Input files

LC/MS data

RMassBank handles high-resolution LC/MS spectra in mzML format in centroid^[The term "centroid" here refers to any kind of data which are not in profile mode, i.e. don't have continuous m/z data. It does not refer to the (mathematical) centroid peak, i.e. the area-weighted mass peak.] or in profile mode.

Data in the examples was acquired using an LTQ Orbitrap XL instrument in profile mode, and converted from profile-mode RAW into centroid-mode mzML using MSConvertGUI from ProteoWizard. The settings were as shown in the screenshot below (note the "Peak Picking" filter.) fig:proteowiz

Fig. 1: ProteoWiz settings for conversion to mzML

In the standard workflow, the file names are used to identify a compound: file names must be in the format xxxxxxxx_1234_xxx.mzXML, where the xxx parts denote anything and the 1234 part denotes the compound ID in the compound list (see below). Advanced and alternative uses can be implemented; consult the implementation of msms_workflow and findMsMsHR for more information.

Compound list

A compound list in CSV format is required to identify all compounds unambiguously. The CSV file is required to have at least the following columns, which are used for further processing and must be named correctly (but present in any order): ID, Name, SMILES, RT, CAS. The columns ID and SMILES must be filled, the other columns must be present in the file but do not need to be filled. ID specifies an (arbitrary) numeric ID code which must be < 4 digits long; SMILES specifies a SMILES code with the chemical structure of the compound (and is used to extract the molecular formula, for calculation of molecular masses, for database searching in CTS etc.) Although the columns Name, RT, CAS have to be present, the information in the columns is only used if the cells are filled. RT, if present, specifies the retention time (in minutes; $\pm$ a window specified in the RMassBank options, see below) where a LC/MS file is searched for the compound spectra. CAS and Name are used as additional information while retrieving annotations from CTS. The compound list doesn't have to be ordered in any particular way. It can contain large numbers of compounds, even compounds which will not be actively used by the script (Note: Unused compounds don't require a SMILES code, since they will not be accessed.)

An example list is provided with the RMassBankData package, and can be copied into a local folder, viewed and edited:

    package="RMassBankData"), "./Compoundlist.csv")


A number of different settings influence RMassBank. They are partly parameters for data processing and partly constants used for annotation.

A settings template file, to be edited by hand, can be generated using


where mysettings.ini is the file that will be generated. This file should then be edited. Important settings are:

See also the manpage ?RmbSettings for a description of all RMassBank settings.

The workflow

Mass spectrometry workflow

In the first part of the workflow, spectra are extracted from the files and processed. In the following example, we will process the narcotics spectra from the RMassBankData package.

For the workflow to work correctly, a settings file (generated as above and edited accordingly) before must be loaded first.


(Note: the template file generated by RmbSettingsTemplate() has no OpenBabel directory specified. Correspondingly, RMassBank will use the CACTUS service instead to generate MOL files. For your actual use, it is strongly recommended to install OpenBabel and specify its install directory in the settings! The CACTUS structures are visually less appealing since they have all hydrogen atoms explicit, and CACTUS is only a backup solution.)

First, create a workspace for the msmsWorkflow:

w <- newMsmsWorkspace()

The full paths of the files must be loaded into the container in the array files:

files <- list.files(system.file("spectra", package="RMassBankData"),
     ".mzML", full.names = TRUE)
# To make the workflow faster here, we use only 2 compounds:
w@files <- files[1:2]

Note the position of the compound IDs in the filenames. Historically, the "pos" at the end was used to denote the polarity; it is obsolete now, but the ID must be terminated with an underscore.

Additionally, the compound list must be loaded using loadList (here, using the formerly copied list from RMassBankData):


This creates a variable compoundList in the global environment, which stores the compound data. Now, we can start the complete workflow to extract [M+H]+ spectral data. The workflow standard workflow consists of 8 steps.

The argument archivename specifies the prefix under which to store the analyzed result files. The argument mode specifies the processing mode: pH (positive H) specifies [M+H]+, pNa specifies [M+Na]+, pM specifies [M]+, mH and mFA specify [M-H]- and [M+FA]-, respectively. (I apologize for the naming of pH which has absolutely nothing to do with chemical pH values.)

Basically, this runs through the entire workflow, which is explained in more detail below: Step 1: using the function findMsMsHR, all the files in files are searched for MS2 spectra of their respective compound. The found spectra are stored in the array specs. Step 2: A molecular formula fit is attempted for every peak, using the molecular formula of the parent compound as limiting formula, using the function analyzeMsMs. The results are stored in the array analyzedSpecs. Step 3: The analyzed spectra from the array analyzedSpecs are aggregated into the list aggregatedSpecs. This uses the function aggregateSpectra. Step 4: Using the function recalibrateSpectra, a recalibration curve is calculated from the peaks in aggregatedSpecs, and all spectra from specs are recalibrated using this curve. The result is stored in recalibratedSpecs. The recalibration curve is stored in rc. Step 5: The recalibrated spectra (recalibratedSpecs) are re-analyzed with analyzeMsMs and the results stored in analyzedRcSpecs. Step 6: The reanalyzed recalibrated spectra are aggregated with aggregateSpectra into aggregatedRcSpecs. Unmatched peaks in aggregatedRcSpecs are cleaned from known electronic noise using cleanElnoise. A backup copy of all present results is saved as archivename``.RData. Step 7: Using reanalyzeFailpeaks, all unmatched peaks from spectra in aggregatedRcSpecs are reanalyzed, allowing $N_2O$ as additional elements (to account for oxidation products and $N_2$ adducts). The results are stored in reanalyzedRcSpecs. A backup copy of all present results is saved as archivename``_RA.RData Step 8: The function filterMultiplicity is applied to the peaks: Peaks which occur only once in all analyzed spectra of a compound are eliminated. The filtered list is stored under refilteredSpecs, and a final version of all results is saved as archivename``_RF.RData. Additionally, filterMultiplicity creates a CSV file with a list of (relatively) high-intensity unassigned peaks with the name archivename``_Failpeaks.csv, which should be manually checked. Peaks to include must be marked with OK = 1.

The steps can be called individually using the steps parameter of msms_workflow. Using the newRecalibration parameter, one can specify if RMassBank should do a new recalibration (default, TRUE) or use the recalibration curve stored in rc (FALSE). This is useful for re-using a recalibration curve in the reanalysis of the same data in another mode: After the detection and processing of all [M+H]+ spectra, which will be present for a large number of compounds, one can rerun the workflow with newRecalibration = F, mode="pNa" and reuse the same calibration curve for Na adduct spectra (which on their own would be too few for a sufficiently good recalibration curve.) The useRtLimit parameter activates or deactivates the usage of retention time constraints when searching for spectra with findMsMsHR.

It is useful to perform the workflow in two blocks, the first being step 1-4 and the second being 5-8. After step 4, a graph is displayed which allows the user to visually evaluate the performance of the recalibration. The top graphs show the distribution of the mass deviation of MS/MS fragments from the predicted mass and the recalibration curve calculated from them; the bottom graphs show the mass deviation of MS precursor ions. The graph to the left is a complete xy plot while the graph to the right is a 2D histogram (if the package gplots is installed on the user's computer).

TODO: Workflow execution in Chunk 10 is currently disabled, I execute Chunk 11 instead for steps that are already done.

w <- msmsWorkflow(w, mode="pH", steps=c(1:4), archivename = 

The recalibration can also be plotted at a later stage:


If you are experimenting with new datasets which might give errors, it is advised to run the workflow step by step. This is because if an error occurs, you will lose all intermediate results from the workflow, which might complicate finding the errors. (E.g., if you process steps 2-4 and an error occurs in step 3, you will lose the results from step 2.)

    w <- msmsWorkflow(w, mode="pH", steps=1)
    w <- msmsWorkflow(w, mode="pH", steps=2)
    w <- msmsWorkflow(w, mode="pH", steps=3)
    # etc.

It can be useful to check if any data is retrieved at step 1:

lapply(w@spectra,function(s) s@found)

To check the progress through the workflow, call e.g.:


Note that usually a recalibration curve should be done which >15 compounds, and it will become smoother with more compounds. To show the curve found with the full dataset, we can load the preprocessed dataset from the RMassBankData package in another workflow container.

# In the really evaluated workflow, we do the following:
# we run steps 1 through 3, load the recalibration curve from a stored workflow
# and recalibrate the data using that curve.
storedW <- loadMsmsWorkspace(system.file("results/pH_narcotics_RF.RData", 

Since this recalibration curve was calculated from a MassBank run of the whole 15 file-dataset, we can copy it into our workspace and use it to recalibrate our data without making a new recalibration curve:

# Just to display the recalibration curve as calculated from
# the complete dataset:
storedW <- msmsWorkflow(storedW, mode="pH", steps=4)
# Copy the recalibration to workspace w and apply it
# (no graph displayed here)
w@rc <- storedW@parent@rc
w@rc.ms1 <- storedW@parent@rc.ms1
w <- msmsWorkflow(w, mode="pH", steps=4, archivename = 
    "pH_narcotics", newRecalibration = FALSE)

The second part of the workflow can then be processed:

w <- msmsWorkflow(w, mode="pH", steps=c(5:8), archivename = 

If the workflow is performed manually, the results can be stored at any time using

archiveResults(w, filename)

where the former writes the results to a file and the latter duplicates the R objects with a prefix in front of their names. (Note that during the whole workflow, the results are stored automatically after steps 6, 7, and 8 if an archivename is given. So the archivename) parameter is only pro forma for the steps 1-5, but can be added for consistency.

Result files from the workflow on the RMassBankData narcotics spectra dataset are included in RMassBankData, including a marked Failpeaks.csv list.

MassBank record workflow

An analyzed spectral dataset can then be processed to produce MassBank records. This is done in two major steps: First, annotations for all compounds are retrieved from the Internet, if they are not already present from previously compiled spectra (e.g. if an Internet annotation has already been used to create a [M+H]+ spectrum, it can be reused in the [M-H]- spectrum automatically.)

First, a workspace for the MassBank results must be created starting from processed msmsWorkflow results, and potential pre-existing infolists must be loaded.

To illustrate the workflow, a half-complete annotation list is included in RMassBankData.

mb <- newMbWorkspace(w)
mb <- resetInfolists(mb)
mb <- loadInfolists(mb, system.file("infolists_incomplete",

Usually, one would call the function with a personal folder:

mb <- resetInfolists(mb)
mb <- loadInfolists(mb, my_folder_with_csv_infolists_inside)

If we checked the Failpeaks.csv from the previous step and found some important peaks we want to add manually, we can do so and load the peaks into the additional_peaks array:

mb <- addPeaks(mb, my_corrected_Failpeaks.csv)

Now, the record generation workflow can be started:

mb <- mbWorkflow(mb, infolist_path="./Narcotics_infolist.csv")

For all the compounds which were not in the infolists in the infolists_incomplete folder, an entry is fetched and written to Narcotics_infolist.csv (if no infolist_path is specified, the default path is ./infolist.csv.) This file should then be edited and fixed by hand. The entries don't have to be complete; mandatory fields are: at least 1 name, the formula, the exact mass, SMILES code, InChI standard code, InChI standard key. Common errors which must be fixed by hand: 2 near-identical names in the infolist; a very high ChemSpider ID where a lower one exists (which is "better"), a ChEBI entry saying "ChEBI" instead of the actual ChEBI code.

CAUTION: At this stage the compound name is taken from the user-provided compound list and one IUPAC entry from CTS. Please check your compound list carefully! The original naming system from CTS will be reinstated once the scoring system is re-included in the new services.

After fixing the CSV infolist, it should be copied into the infolist folder and the infolist reloaded:

mb <- resetInfolists(mb)
mb <- loadInfolists(mb, my_folder_with_csv_infolists_inside)

For simplicity / easy testing, a full list for the narcotics dataset is included in RMassBankData:

mb <- resetInfolists(mb)
mb <- loadInfolists(mb, system.file("infolists", package="RMassBankData"))

When we run the workflow again, the line "no new data added" means that the infolists were complete and the workflow can therefore continue.

mb <- mbWorkflow(mb)

The workflow goes through the following steps: Step 1: For compound IDs not in a loaded infolist, new data is fetched from the CTS using the function gather.data and stored in mbdata in tree-like format. Step 2: If new data was retrieved, it is exported to the infolist_path in flat-table format and the workflow stops, otherwise the workflow continues. Step 3: The infolists loaded with loadInfolists are transformed into tree-like MassBank compound information with readMbdata and stored as mbdata_relisted. Step 4: Using the function compileRecords, the compound information from mbdata_relisted is combined with the spectral data and peak lists from aggregatedRcSpecs and refilteredRcSpecs to create compiled records (stored in compiled). All compiled records with at least one good spectrum per compound are in compiled_ok. Step 5: The function toMassbank converts the records into text-file arrays, stored in mbfiles. Step 6: Molfiles are generated for all compounds using createMolfile and stored in molfiles. Step 7: The data stored in the R variables mbfiles and molfiles is written to physical files using exportMassbank in a subfolder named after the MassBank entry prefix. Step 8: A list.tsv file is created using makeMollist.

Subsequently, the two folders moldata and recdata can be zipped and uploaded. This wasn't automated because the Windows version of zip needs additional installed tools.

Note: here, step 6 uses molfile data generated by CACTUS. As stated above, it is strongly recommended to install OpenBabel and add its path to the configuration file for use in mbWorkflow step 6.

Session information


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RMassBank documentation built on Nov. 8, 2020, 6:06 p.m.