Mass Profiler Pro Quick Start - Chemical Analysis, Life ... Profiler Professional Quick Start Guide...

Agilent G3835AA MassHunter Mass Profiler Professional Software

Quick Start Guide

What is the Mass Profiler Professional B.02.00?

Mass Profiler Professional (MPP) is a powerful visualization and analysis solution designed for use with mass spectrometry (MS) data in metabolomics and proteomics. It allows you to display and analyze large data sets on your personal computer. MPP helps you explore relationships in GC/MS or LC/MS data, regardless of whether peaks are identified or are unknown.

How do I get started with Mass Profiler Professional B.02.00?

After installation of MPP, you can get started immediately since it comes preloaded with a demonstration experiment to show you the functionality of the product. A project called “Malaria” contains an experiment called “Malaria LCMS pH 7 ESI+.” We encourage you to explore this demonstration project to get to know Mass Profiler Professional. Use the familiarization tutorial that begins on the next page for your first steps with the software.

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Familiarization tutorial: How can I do a new analysis?

Exercise 1. Begin creating a new experiment

An easy way to start using the software is to recreate the sample experiment from the demonstration project. The original data you need is provided with the installation. The data files are located in the samples directory of the main installation folder, usually found in C:\Program Files\Agilent\MassProfilerPro.

Follow the steps below to create a new experiment with the provided data.

Steps Detailed Instructions Comments

1 Start Mass Profiler Professional.

2 In the Startup dialog box, create or open a project.

If you want to use the Malaria project:a Select Open recent project.b Click the down-arrow and select the

Malaria project.

If you instead want to create a new project, select Create new project.

• Open recent project opens the project and the experiment that was stored in this project.

• A project is a container for a collection of experiments. A project can have multiple experiments on different sample types and organisms.

2 Mass Profiler Professional Quick Start Guide

3 Create a new experiment. a Do one of the following:• Click the New

Experiment icon on the Toolbar.

• In the Project menu, click New Experiment.

b Verify that you see the New Experiment dialog box shown below.

• The New Experiment wizard guides you through the creation of the experiment.


Mass Profiler Professional Quick Start Guide 3

4 Fill out the New Experiment dialog box.

a In the Experiment name box, type an appropriate name for the new experiment.

b For Experiment type, do one of the following:• If you are using the Malaria data set

that is supplied with MPP, select Unidentified.

• If you are using your own data set, select an Experiment type that is consistent with that data. Click Help for further guidance.

c For Organism, do one of the following:• If you are using the Malaria data set

that is supplied with MPP, select Homo sapiens.

• If you are using your own data set, select the appropriate organism or choose Not Applicable. Note that selecting an organism is most important if and when you choose to use the pathway analysis features of MPP.

d For Workflow type, read the comments to the right, then select Guided Workflow.

e Click OK.

• MPP has two modes for experiment creation: Guided Workflow and Advanced Analysis.

• The Guided Workflow provides you with full guidance and is a good starting point.



5 Choose the data source. a In the dialog box for MS Experiment Creation Wizard (Step 1 of 10), do one of the following:• If you are using the Malaria data set

that is supplied with MPP, click MassHunter Qual as the data source.

• If you are using your own data set, click the source of the sample files that you will import into MPP.

b Click Next>>.

• To control the guided workflows:• Click the Next>> button to go

to the next step. • Click the

<<Back button to go back and make modifications to your settings.



6 Import new data files, or use previously loaded data.

For this exercise, import new data files: a Click Select Data Files to display the

file selection dialog. b The dialog will most likely already

point to the sample directory in the main Mass Profiler Professional installation directory, but if not, browse to C:\Program Files\Agilent\ MassProfilerPro\samples\Malaria.

c Select all the .cef files you want to load and click OK:• 1-1_pH7_pos_01.cef• 1-2_pH7_pos_01.cef• 1-3_pH7_pos_01.cef• 1-4_pH7_pos_01.cef• 3-1_pH7_pos_01.cef• 3-2_pH7_pos_01.cef• 3-3_pH7_pos_01.cef• 3-4_pH7_pos_01.cef

d Click Next>> to complete the selection of the data files.



7 Adjust filters for the data you load into MPP.

a In the dialog box for MS Experiment Creation Wizard (Step 6 of 10), exclude or include compounds found in the input data files.

b When you are finished adjusting the available filters, click Next>>.



8 Adjust alignment parameters. a In the dialog box for MS Experiment Creation Wizard (Step 7 of 10), set the alignment parameters.

b When you are done, click Next>>.

• Alignment parameters are used by MPP to align and group compounds by similar masses and retention times across multiple sample files.



9 Examine the results of the alignment.

a In the dialog box for MS Experiment Creation Wizard (Step 8 of 10), view the results of the alignment.

b When you are done, click Next>>.

• The unidentified compounds are aligned and the software displays summary data from the alignment.

• The summary includes a Mass vs. RT plot and a count of how many of the compounds found in the entire data set were actually found within each sample.



10 Normalize your data using an internal standard and/or an external scalar.

a In the dialog box for MS Experiment Creation Wizard (Step 9 of 10), do one of the following:• If you are using the Malaria data set

that is supplied with MPP, skip the normalization step.

• If you are using your own data set, set the values under both the Internal Standard and External Scalar tabs.

b Click Next>>.

11 Choose a baselining option that is appropriate for your data.

a In the dialog box for MS Experiment Creation Wizard (Step 10 of 10), do one of the following:• If you are using the Malaria data set

that is supplied with MPP, choose Baseline each entity to median across samples.

• If you are using your own data set, read the comments to the right, then click the appropriate setting.

b Click Finish.

• Use Z-Transform when you compare data from different sources, or Baseline each entity to median across samples to treat all compounds equally regardless of their intensity.

• After you click Finish, the application loads the files; performs the alignment, normalization, and baselining; and then creates a new experiment in the project that is currently open.

12 Continue to set up the experiment, using either the Guided Workflow or the Advanced Workflow.

Do one of the following, depending on your choice of workflow in step 4 on page 4:

• If you chose the Advanced Workflow, you are done creating the experiment and now can use the Workflow Browser on the right side of the application to proceed through the various steps to set up the experiment and perform the analysis.

• If you chose Guided Workflow, proceed to the next exercise.



Exercise 2. Continue with the Guided Workflow

In this exercise, you proceed through the dialog boxes that the Guided Workflow automatically presents. The Guided Workflow executes a data import wizard followed by a differential expression wizard.


1 View the Summary Report. a Verify that you see the Guided Workflow wizard, as shown below.

b View the profile plot.c Click Next>>.

• The Guided Workflow wizard shows a profile plot and a series of steps to finish the experiment setup and perform analysis.

• It displays a sequence of steps on the left, and highlights the current step.


2 Set up the Experimental Grouping. If you are using the Malaria data set that is supplied with MPP:a In the dialog box, click the

Load Experiment Parameters from File button.

b In the file selection dialog box, navigate to the same directory from which you selected the sample files in step 6 on page 6.

c Select the tsv file named MALARIA EXPERIMENT PARAMETERS and click Open.

d When you are done, verify that your dialog box looks like the one below.

e Click Next>>.

If you are using your own data set:a Click the Add Parameter... button to

set up a parameter and enter the values for each data file.

b Click Next>>.

• In Experiment Grouping, samples with same parameter values are treated as replicates.

• In the Malaria example, you specify which files contain data from infected samples and which contain data from uninfected samples.



3 Set filter parameters for the Filter Flags.

a If desired, click Re-run Filter and specify that at least 2 out of 8 samples have acceptable values.

b When you are done adjusting parameters, click Next>>.

• The Filter Flags wizard page allows you to filter the input data based upon Present, Marginal, or Absent flags that may be in the data files themselves, and by declaring that a particular compound must appear a minimum number of times across all samples.



4 Set filter parameters for Filter by Frequency.

a If desired, click Re-run Filter to adjust the current parameters.

b When you are done adjusting this filter, click Next>>.

• The Filter Frequency wizard page allows you to filter input data based upon the frequency with which any compound appears in each sample in the experiment.



5 Use QC on samples to assess sample quality.

Do one of the following:• For the Malaria data set, simply click

Next>>.• For your own data set, use this

opportunity to review the data and remove any ambiguous samples before you begin the data analysis.

• Use Ctrl-drag to rotate or Shift-drag to zoom the 3D PCA plot.

• The QC on Samples wizard page shows a principal component analysis (PCA) plot to help verify the quality of the samples.

• This plot often shows you if any samples have exceptionally different values from the rest, or if a sample was assigned an incorrect parameter value.



6 Adjust parameters for Significance Analysis and Fold Change.

a If desired, click Re-run Analysis to adjust the current parameters.

b When you are done adjusting these parameters, click Next>>.

• In Significance Analysis, entities are filtered based on their p-values calculated from statistical analysis.

• The entities that satisfy the Significance Analysis are passed on for the Fold Change Analysis.

• The Significance Analysis wizard page allows you to adjust the cutoff values for either the p-value or fold change.

• MPP is showing the fold change of the abundance value for each compound across the various samples in the experiment.

• Set the filter so that you focus on the compounds that have intensity values that are most significant among the samples in the experiment.



7 Start the IDBrowser Identification.

NOTE: If you are working with the Malaria data set, the Agilent METLIN Personal MetaboliteDatabase must be installed to complete this portion of the exercise. Please be sure METLIN is installed before continuing.

a Click the IDBrowser Identification button that appears just above the table of data.

b Go on to the next exercise.

• In the IDBrowser Identification wizard page, the table shows the compounds that remain after all of the previous filtering steps.

• The compounds are identified only by the Mass@RT values that are shown in the Compound column in the table.

• You use the ID Browser to help identify the compounds in that list.



Exercise 3. Use the MassHunter ID Browser to identify the compounds

In this exercise, you use the ID Browser to identify the compounds from the last step of the previous exercise.


1 Select the identification methods for the compounds.

In the first Compound Identification Wizard dialog box, do one of the following:• If you are using the Malaria data set,

click Next>>.• If you are using your own data set,

select the desired identification methods, then click Next>>.

• When you launch the ID Browser, it receives the list of entities (compounds) from the last step of the previous exercise.

• The ID Browser attempts to identify those compounds using the criteria on this wizard page and the next one.


2 Set parameters for the identification techniques.

In the second Compound Identification Wizard dialog box, do one of the following:• If you are using the Malaria data set,

click Next>>.• If you are using your own data set,

change the settings for the identification algorithms as needed, then click Finish.

• Using the ID Browser, there are many different ways to adjust the identification algorithms.

• Click the Help button to get a full description of all available options and features.



3 View and adjust the identifications. In the Agilent MassHunter ID Browser, do one of the following:• If you are using the Malaria data set,

view the results but make no adjustments.

• If you are using your own data set, review and change the identifications if necessary.

• The ID Browser shows you information about each identified compound.

• It also gives you the ability to make adjustments to the identifications.

• Click the Help menu to get more information.



4 Accept the current identifications and return to Mass Profiler Professional.

• In the Agilent MassHunter ID Browser, click File > Save and Return.

• The ID Browser sends all the information it has generated back to MPP.

5 Examine the results in Mass Profiler Professional.

a Notice that many of the compounds in the table have been identified.

b Click Finish.

• When you return to MPP, the Compound column is updated.



At this point, you have created a new experiment and we invite you to explore all the functionality of the product. We recommend that you consult the manual (Help menu) and view online tutorials on the Agilent Web site.

6 View the profile plot. • When the wizard is complete, the software displays a profile plot.

• The navigator on the left has results from each step in the wizard, so you can retrace the experiment.

• Each entity list can be used in any of the analysis functions available in MPP.

• The result of the analysis is presented hierarchically, making it easy to see the steps that were done in the guided workflow.



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© Agilent Technologies, Inc. 2009

Printed in USA First edition, September 2009

*G3835-90000*G3835-90000

www.agilent.com

In this book

The Quick Start Guide presents first steps to use the MassHunter Mass Profiler Professional Software

If you have comments about this guide, please send an e-mail to [email protected].

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Mass Profiler Pro Quick Start - Chemical Analysis, Life ... Profiler Professional Quick Start Guide...

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