Multi-Q 2 Quick Start
In Quick Start, we demonstrate the processes to directly load existing quantitation results and run Multi-Q 2 with minimum parameter settings (Section 1), and perform quantitation tasks on two data sets, “Gatto_TMT6”
(Section 2) and “Hultin_iTRAQ8” (Section 3).
Users need to first download Multi-Q 2 and extract the compressed file, put the folder of Multi-Q 2 in a desired location, for example, the Windows desktop. Start Multi-Q 2 by double-clicking Multi-Q_v2.exe in the folder.
Section 1. Loading existing quantitation results directly
To skip all the parameter settings and try out Multi-Q 2 in the most convenient fashion, we provide the following steps and necessary files so that users can directly load existing quantitation results of a sample data set and explore various functions in Multi-Q 2.
Download “project_TMT6.7z” from our website (http://ms.iis.sinica.edu.tw/COmics/Software_Multi- Q2.html) and extract the file in “D:/”. The 7z file consists of a folder, which is a project folder named
“project_TMT6”, and its subfolder “Exp_GattoTMT6” is an experiment folder containing the binary files for the experiment.
In the Multi-Q 2 main window, right-click the Project List in Project Explorer and select “Add Existing Project”.
As shown in the figure below, a file browser dialog will pop up. Find the file “project_TMT6.multiq” in folder
“D:/project_TMT6/” and open it. The corresponding project node and experiment node will show up in Project Explorer.
Double-click the experiment node, or right-click the experiment node and select “View”, as shown below, to load the quantitation results into Multi-Q 2.
After the quantitation results are loaded, the protein, peptide and PSM tables display the ratios for proteins, peptides, and PSMs, respectively. Users can click on different proteins to navigate their peptide and PSM ratios. All the functional buttons are enabled. Their functions are described in details in Multi-Q 2 Manual.
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The next two sections show step-by-step procedures for quantitation from scratch.
Section 2. Perform Quantitation for Gatto-TMT6 data set
Download the file “Gatto-TMT6.7z” from our website and extract the file in “D:/”. The data set “Gatto-TMT6”
is from a TMT-6 experiment on Erwinia caratovora. The 7z file contains one spectrum file in centroid mode, one pep.xml file as the output of iProphet (peptide-level validation result), and one csv file as the output of Mayu (protein-level validation result).
2.1 Using Project Wizard to initialize a project for quantitation
When executing Multi-Q 2, first, in Project Explorer of the main window, click the New Project button or right-click on the Project List and choose New Project, as shown in the figure below. The Project Wizard form will pop up.
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In Project Wizard, perform the following two steps to initialize a project:
Step 1: Type the project name and choose the file location for the project, as shown in the figure below.
Name the project as “project_TMT6” and choose “D:\” as the project location. Project is a folder which may consist of several subfolders, each of which corresponds to an experiment that has its own data set, parameter settings, quantitation algorithms etc. Click on “Finish” when it is done.
Step 2: input project description (optional). Click on “Finish” when it is done.
2.2 Using the Quantitation Wizard to create a new experiment in the project
In the main window of Multi-Q 2, click the New Experiment button or right-click on the project node in Project List and select New Experiment. The Quantitation Wizard will pop up.
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Users can perform the following steps in the Quantitation Wizard to specify the input information of the experiment and to perform quantitation:
Step 1: Set the parameters.
Note that parameters in red boxes are data set dependent and need to be adjusted accordingly. Please set the parameters according to the figure below. The rest are algorithmic parameters which can be used directly for now, and they can be easily modified after the quantitation is finished (more details in Section 3.4 of Multi-Q 2 User Manual).
The parameter “Experiment name”, Exp_GattoTMT6, is used to name a subfolder in the project folder, which will be generated after the quantitation is done. In “MS Spectra File Information”, because there is only one spectrum file in our sample data, the Sample number, Fraction number and Replicate number should all be 1.
The parameters in Peak Extraction Parameters are described in details in Section 3.1 of Multi-Q 2 User Manual. Once the parameter setting is completed, click “Next >” and move on to the next step.
Step 2: Specify input data files for Multi-Q 2, as shown in the figure below. For this example, a spectrum file and two search result files need to be included.
Click to add the only spectrum file in mzXML format. Click to add the csv file (protein-level validation result) and the pep.xml file (peptide-level validation result) downloaded from our website. The files will appear in the left panel. Then, drag the files one-by-one from the left panel to the corresponding nodes in the right panel, as shown in the following figure.
Specifically, to obtain the data for quantitation, drag
“TMT_Erwinia_1uLSike_Top10HCD_isol2_45stepped_60min_01.mzXML” onto the node “Replicate 1”,
“20161213_Comet+XTandem_Mayu.csv” onto the node “Search Result at Protein Level”, and
“20161212_Comet+XTandem_iProphet.pep.xml” onto the node “Search Result at Peptide Level”.
Click “Next >” when these steps are done.
Step 3: Specify the labeling method and define ratios to be calculated.
For this example, select “TMT-6plex” in the Labeling Method. By default, five ratios for TMT-6plex are predefined as taking the first channel as the denominator and the other channels as the numerators. The five predefined ratios will be shown in the list box in the center, as illustrated in the figure below.
Click “Next >” and move on to the final step.
Step 4: Double-check input information for quantitation.
An overview of all the parameter settings will be shown to users for confirmation. If confirmed, click “Next
>” to start the quantitation.
Step 5: Multi-Q 2 starts to process the data.
Quantitation consists of three steps: reading search result, reading raw data, and performing quantitation.
A check mark will appear in front of each row if the operation is processed successfully.
After all the tasks are completed ( showing up for all the operations), click “Finish”.
2.3 Viewing quantitation results in the main window of Multi-Q 2
After finishing quantitation, Multi-Q 2 will present the quantitation results in the Quantitation Result page (right-hand side of the main window), as shown below.
Correspondingly, Project Explorer at top left of the main window shows the hierarchical structure of the quantitation projects. The project “project_TMT6” (highlighted by a red box) and the experiment
“Exp_GattoTMT6” (highlighted by a blue box), which were previously specified by using the Project Wizard are listed in the figure below.
Project “project_TMT6” corresponds to the folder “D:/project_TMT6”; experiment “Exp_GattoTMT6”
corresponds to the folder “D:/ project_TMT6/ Exp_GattoTMT6”, as illustrated in the figure below.
The experiment folder contains the binary files of the data sets that allows rapid loading of configuration settings and quantitation results without the need to read the original files in text format. This design is useful when users intend to switch quantitation algorithms or parameters and re-quantify the entire data set.
Section 3. Perform Quantitation for Hultin-iTRAQ8 data set
Download the file “Hultin-iTRAQ8.7z” from our website and extract the file in “D:/”. The data set
“Hultin_iTRAQ8” is from an iTRAQ-8 experiment on A549 cell line. The 7z file contains five centroided spectrum files from different fractions, one pep.xml file from the output of iProphet (peptide-level validation result), and one csv file from the output of Mayu (protein-level validation result).
Most of the steps are similar to those for analyzing the Gatto_TMT6 data set.
3.1 Using the Project Wizard to initialize a project for quantitation Create a project named “project_iTRAQ8” in Project Explorer.
3.2 Using the Quantitation Wizard to create a new experiment in the project
Create a new experiment under project_iTRAQ8 with Quantitation Wizard. Perform the following steps:
Step 1: Set the parameters according to the figure below.
Experiment is named as Exp_Hultin_iTRAQ8. In MS Spectra File Information, because the data set consists of five fractions, Fraction number is specified as 5.
Settings in Algorithmic Parameters should be set as follows: WeightedPepRatio for “Protein ratio calculation method”, WeightedPsmRatio for “Peptide ratio calculation method”, True for “Enable impurity correction”, and False for “Enable ratio compression correction”. Normalization is applied at peptide level, i.e., true for
“Peptide ratio-based normalization” and false for the rest of normalization methods.
This algorithmic combination yields the best quantitation accuracy in terms of AUCCD (Area Under the Curve of Coverage vs Deviation), AER (Average Error Rate), and RMSE (Root Mean Square Error). For more details please refer to our manuscript. Click “Next >” and move on to the next step.
Step 2: Specify input files for Multi-Q 2. s
The figure below shows the form for adding spectrum files and search result files for quantitation in Multi-Q 2.
Click to add the spectrum files (five spectrum files for Hultin_iTRAQ8 data set) in mzML format. Click to add the csv file and the pep.xml file downloaded from our website, which are “2017-06- 22_16.10.04_psm_protFDR0.01_t_1.08.csv” and “interact.iProphet.pep.xml”, respectively. The files will appear in the left panel, as shown below.
Drag “interact.iProphet.pep.xml” onto the node “Search Result in Peptide Level”,
“2017-06-22_16.10.04_psm_protFDR0.01_t_1.08.csv” onto the node “Search Result in Protein Level”.
Note that it is not required to drag the five mzML files to the corresponding nodes in the right panel. Simply click “Next >” and all the mzML files will be automatically included by Multi-Q 2, as shown below.
Step 3: Specify the labeling method and define ratios to be calculated.
Select “iTRAQ-8plex” in the Labeling Method. By default, the seven predefined ratios for iTRAQ-8plex will be displayed in the list box at the center, as illustrated in the figure below. Click “Next >” and move on to the final step.
Step 4: Double-check input information for quantitation.
If all settings are confirmed, click “Next >” to start the quantitation.
Step 5: Multi-Q 2 starts to process the data.
After all the tasks are completed, as shown below, click “Finish”.
3.3 Viewing quantitation results in the main window of Multi-Q 2
Multi-Q 2 displays the quantification results in its main window, as shown below. In Project Explorer, the experiment “Exp_Hultin_iTRAQ8” appears as a child node of “project_iTRAQ8”.
The corresponding experiment folder and binary files are also created. Users can launch file explorer to check them, as illustrated below.
