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Tutorial

This tutorial will guide you through all the steps needed to (i) detect the AAS in MS mode and (ii) to confirm the identity of the compound by MS/MS using the MLibrary tool. Moreover, it explains how to construct both MS and MS/MS databases.

 

Contents

  1. Detecting AAS in single MS mode

    1. Load experimental data

    2. Perform MLibrary MS analysis

  2. Confirming the identity of AAS in MS/MS mode

    1. Load experimental data

    2. Perform “MS/MS Library Analysis”

    3. Perform “MS/MS Std. Match Analysis”

    4. Perform “MS/MS Marker Analysis”

  3. Construction of MLibrary databases

    1. MLibrary MS Database creation

    2. MLibrary MS/MS Database creation

    3. MLibrary MS/MS Marker Database creation

    4. MLibrary Modifications creation

 

Detecting AAS in single MS mode 

1. Load experimental data

Press the "Load MS Data" button on the toolbar.

Load MS Data

A dialog box will appear:

Load MS Data

You have to provide the following information:

  • Peak intensity: this value acts as a peak filter, avoiding the load of peaks with intensity lower than this parameter value.
  • Data File: A text-plain CSV file containing the experimental data. You can download one example here.

 

After the experimental data is loaded you can see the spectrum and the peak list with the correspondent intensity on the screen.

MS Data


 

2. Perform MLibrary MS analysis

Press the "MS Analysis" button on the toolbar.

MS Analysis

A dialog box will appear:

MS Analysis Dialog

You have to provide the following information:

  • Database: this parameter allows the user to select a specific database.
  • Compound state: this parameter takes into account if the compound is in its “conjugated” or “free” form.
  • Modification: this parameter allows the user to select a known modification, for instance, a modification arriving from the employment of a derivatisation agent.
  • Mass tolerance: the maximum mass error between the database and the experimental mass values.

 

After the "MS Analysis" is performed, the search retrieves the mass values that matched between the experimental data and the database values, showing both experimental and theoretic values as well as the name of the compound and the experimental peak intensity.

MS Analysis

MLibrary also provides an additional operation named “MS Full Analysis”, which performs a search within all the MS databases present in the MLibrary repository.


Confirming the identity of AAS in MS/MS mode

1. Load experimental data

Press the "Load MS/MS Data" on the toolbar.

Load MS/MS Data

A dialog box will appear:

Load MS/MS Data
Likewise for the MS mode, in the MS/MS mode you have to provide the following information:
  • Peak intensity: this value acts as a peak filter, avoiding the load of peaks with intensity lower than this parameter value.
  • Data File: A text-plain CSV file containing the experimental data. You can download one example here.

 

After the experimental data is loaded you can see the spectrum and the peak list with the correspondent intensity on the screen.

 

MS/MS Data

 

 

2. Perform the "MS/MS Library Analysis"

Press the "MS/MS Library Analysis" button on the toolbar.

MS/MS Library Analysis

A dialog box will appear:

MS/MS Library Analysis

 

You have to provide the following information:

  • Compound mass: input the precursor ion mass (optional parameter).
  • Database: this parameter allows the user to select a specific database.
  • Compound: in this field the user has to select the name of compound in the database to which he pretends to compare the MS/MS experimental data.
  • Compound state: this parameter takes into account if the compound is in its “conjugated” or “free” form.
  • Modification: this parameter allows the user to select a known modification, for instance, a modification arriving from the employment of a derivatisation agent.
  • Mass tolerance: the maximum mass error between the database and the experimental mass values.
  • Database mass presence: if the database contains more than one spectrum, this parameter allows the user to select the percentage of presence of a specific peak, within all database spectra, to be considered for the MS/MS analysis.
  • Database mass tolerance: if the database contains more than one spectrum, this parameter represents the maximum mass error between the spectra within the database, to be considered for the MS/MS analysis.

 

After the "MS/MS Library Analysis" is performed, the search retrieves the number of matched peaks and a percentage score of matches within the most intense mass values of the experimental data and the database. For instance, the user may limit the search to the 10 most intense peaks within the inputted file and the library data. This tool is essential to avoid misinterpretation of the results due to the fact that different spectra of the same sample, generally present distinct overall number of mass peaks.

MS/MS Spectra Comparison

 

 

3. Perform "MS/MS Std. Match Analysis"

Another important tool within the MLibrary software is the “MS/MS Std. Match Analysis” operation. With this tool the user can compare two experimental spectra, which is extremely important if we are working at different conditions than the one recorded in the MLibrary MS/MS database.

 

Press the "MS/MS Std. Match Analysis " button on the toolbar.

MS/MS Std. Match Analysis

A dialog box will appear:

MS/MS Std. Match Analysis

 

 You have to provide the following information:

  • Compound mass: input the precursor ion mass (optional parameter).
  • Standard match file: this parameter allows the user to select a specific experimental spectrum file, normally from a standard solution, to compare against the target sample experimental data.
  • Peak intensity: this value acts as a peak filter for the standard match file, avoiding the load of peaks with intensity lower than this parameter value.
  • Mass tolerance: the maximum mass error between the standard match file and the experimental mass values.


After the "MS/MS Std. Match Analysis" is performed, the search retrieves the number of matched peaks and a percentage score of matches within the most intense mass values of the experimental data and the database. The results appear in the same way that showed for the "MS/MS Library Analysis".

 

4. Perform "MS/MS Marker Analysis"

The “MS/MS Marker Analysis” operation allows the user to locate concrete biomarkers into the loaded MS/MS data. This feature is particularly important for the analysis of isobaric compounds having very similar MS/MS fragments or compounds having poor fragmentation pattern. For isobaric compounds the MS/MS Library analysis does not allow the differentiation between the two species, since it will retrieve very close results. For this reason, the identification of exclusive fragments corresponding to each compound is essential for the interpretation of the spectra and it will become essential for their identification.

 

To start press the "MS/MS Marker Analysis" button on the toolbar.

MS/MS Marker Analysis

A dialog box will appear:

MS/MS Marker Analysis
 

You have to provide the following information:

  • Compound mass: input the precursor ion mass (optional parameter).
  • Marker: the user has to select the compound name of the precursor ion, within the markers database.
  • Mass tolerance: the maximum mass error between the markers values and the experimental mass values.

 

After the "MS/MS Marker Analysis" is performed, the search retrieves the number of matched peaks between the experimental data and the biomarkers of the selected AAS.

 

MS/MS Marker Analysis

 

Additionally, the user can use the “MS/MS Full Marker Analysis” in order to search for all the biomarkers stored in the MLibrary repository.

 

Construction of MLibrary databases

The MLibrary compound database is stored in a XML file, so it can be manually modified to include new compounds and markers. You can find your MLibrary database file (mlibrary.xml) inside the "db" folder, located in your MLibrary installation folder. You can download an example database file from here.

 

1. MLibrary MS Database creation

Each MLibrary database file can contains several MS Databases, containing different compounds. You can create a new MS Database adding a "database" tag with a name attribute, as show below.

<database name="Anabolic Androgenic Steroids">
<!-- Compouns will be here -->
</database>

The compounds included in a MS Database must have a name and an associated peak. Compounds are added using a "compound" tag which must include a "peak" tag. As will be further explained, compounds can also contain several associated MS/MS spectra.

<database name="Anabolic Androgenic Steroids">
<compound name="17-a-methyltestosterone">
<peak mass="302.2246"/>
</compound>
<compound name="17-ß-hydroxy-17-methylandrosta-1,4-dien-3-one">
<peak mass="300.2089 "/>
</compound>
<!-- Any number of compounds can be added -->
</database>

2. MLibrary MS/MS Database creation

MS/MS spectra information must be added associated with an existing compound. This is done by adding an “msms” tag in the corresponding compound. An “msms” tag can include any number of MS/MS spectra using the “msmsSpectra” tag. Each MS/MS spectra must have a name, a state (FREE or CONJUGATED) and a modification existing in the MLibrary database (see “4. MLibrary Modifications creation”). Additionally, “msmsSpectra” can include any number of MS/MS spectra.

<database name="Anabolic Androgenic Steroids">
<compound name="17-a-methyltestosterone">
<peak mass="302.2246"/>
<msms>
<msmsSpectra name="17-a-methyltestosterone" state="FREE" modification="Girard T">
<spectrum name="Sample 1">
<peak mass="79.110" intensity="1.48" />
<peak mass="91.117" intensity="2.06" />
<peak mass="93.126" intensity="1.7" />
<peak mass="100.138" intensity="2.9" />
<peak mass="105.133" intensity="1.37" />
<peak mass="255.323" intensity="1.26" />
<peak mass="280.298" intensity="1.43" />
<peak mass="324.349" intensity="2.62" />
</spectrum>
<spectrum name="Sample 2">
<peak mass="73.138" intensity="1.23" />
<peak mass="81.120" intensity="1.44" />
<peak mass="91.123" intensity="1.36" />
<peak mass="93.134" intensity="1.26" />
<peak mass="100.128" intensity="2.7" />
<peak mass="107.150" intensity="1.44" />
<peak mass="117.159" intensity="2.03" />
<peak mass="135.179" intensity="1.33" />
<peak mass="255.313" intensity="2.91" />
<peak mass="273.339" intensity="1.94" />
<peak mass="280.308" intensity="1.99" />
</spectrum>
</msmsSpectra>
</msms>
</compound>
<compound name="17-ß-hydroxy-17-methylandrosta-1,4-dien-3-one">
<peak mass="300.2089 "/>
</compound>
</database>

As can be seen, each MS/MS spectrum has a name and contains several peaks.


3. MLibrary Markers Database creation

A MLibrary database can include a Marker Database, containing a set of markers characterized by one or more masses. The Marker Database is contained inside the “markers” tag, which has several markers (“marker” tag) with a name and that contains a set of masses.  Each mass is declared using a “mass” tag with a value attribute.

<markers>
<marker name="Marker 1">
<mass value="100"/>
</marker>
<marker name="Marker 2">
<mass value="100"/>
<mass value="225"/>
<mass value="250"/>
<mass value="300"/>
</marker>
</markers>

4. MLibrary Modifications creation

A MLibrary database must contain a “modifications” tag containing all the available modifications. Each modification is added using a “modification” tag with the attributes “name” and “variation”. Attribute “name” must contain a unique identifier for the modification, and “variation” is the mass variation associated with the modification. The attribute “modification” of the “msmsSpectra” tag must contain the name of one existing modification.

<modifications>
<modification name="None" variation="0" />
<modification name="Girard T" variation="114.1031" />
</modifications>