User Manual Quantitation
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Phenyx Quantitation Module
This page describes the use of the Phenyx Quantitation Module.
What is the Phenyx Quantitation Module?
The Phenyx Quantitation Module is a post-identification feature dedicated to quantitation of proteins. The module is considered as a post-processing event to one or more identification jobs. It is aimed at covering the following main goals:
- Support a variety of experimental workflows, such as
- Isobaric tags (iTRAQ, Tandem Mass Tag, ...)
- Label-free via spectral counting
- Label-free based on the intensity of the peptide features is available as a combined use of a third party tool such as MSight or Progenesis LCMS
- Support a variety of experimental complexities including multiple biological states and replicates. They can be defined from one or multiple identification runs (therefore also multiple LC-MS runs). Biological or technical replicates can be defined within a job (intra-run) and across jobs (inter-run).
- Implement a generic quantitation algorithm based on averages of peptide intensity ratios (similar to Libra)
- Its Graphical Interface is providing access to configuration and results at various levels of granularity
Current status of the Phenyx Quantitation Module
In the current version of Phenyx 2.6.2, the module supports isobaric tags and spectral counting. The analysis allows multiple replicates (inter-jobs and intra-jobs) and the default algorithms is the one based on averages of peptide intensity ratios (similar to Libra)
How to perform quantitation with Phenyx?
Quantitation is performed as an identification post processing event. The process is therefore called from one or more identification results. In general terms, the steps are the following:
- Select the job(s): In the Phenyx Desktop, choose the job(s) on which quantitation has to be performed (click on check boxes, the job is highlighted in blue)
- Select the quantitation approach: Choose the appropriate quantitation approach (Isobaric Labeling or Spectral Count) item in the "Action on Selected" drop-down menu
- Select the quantitation method: If you selected Isobaric Labeling, select the quantitation method of choice (or configure a new one) in the Quantitation Method Selection Page and click on "Move to Experimental Design Page". If you selected spectral count, you are skipping this step.
- Describe your experimental design: In the Quantitation Experimental Design page, prepare the experimental description of the quantitation: biological states, replicates, ratios to calculate, and click on "submit"
- Evaluate the results: The results are made available in the Quantitation results page and can be exported fronm there as well.
Select the quantitation method (isobaric labeling only)
You can select a quantitation method in the Quantitation Method Selection Page. A method is a set of parameters that describes what reporter ions are to be considered and what isotopic purity correction to apply.
- Choose one of the available methods. The details of the method is appearing in a xml format.
- Select Move to Experimental Design Page" to be redirected to the next step
Note: The method files can be edited by a Phenyx administrator. The corresponding files are locted in a quanti subdirectory of the default user directory. User restricted methods can also be stored in a quanti subdirectory of any user directory.
Define the experimental design (isobaric labeling)
You can define the experimental design of the analysis in the Quantitation Experimental Design page:
- Define the number of experimental biological states to compare
- Attribute a custom name to each State by clicking on "State n", edit the label. The attributed label will appear as tooltip in the results page
- You can also attribute custom names to the job items (these are the inter-job replicates names). The attributed label will appear as tooltip in the results page
- Drag-and-drop the quantitation labels (reporter ions, etc) in the appropriate states for each jobs; move any useless or unused quantitation label to the 'Unused Labels' field. You can leave multiple labels in a single state, in the case of intra-job replicates
- Define the number of Ratios to be calculated and define Nominator and Denominator for each of them
- Go to the Filters page and select what additional filters you want to attribute to the calculation.
- Minimum Number of Peptides is the minimum number of peptide matches to report a protein
- Min z-Score Value is the minimum z-Score that a peptide match must reach to be quantified
- Max p-Value is the highest p-Value that a peptide must reach to be quantified
- Min Intensity Value is the minimum signal intensity that a reporter ion must reach to allow quantitation of the corresponding peptide. Note: This value might have very variable thresholds according to the instrument type and signal processing option. It is recommended to open one spectrum match detail to evaluate the scale.
- Click on the 'Submit' button (top panel). A progress bar is shown, and the result page appear in a new window. The calculation might take a few seconds to a few minutes, depending on the size of the identification results.
Define the experimental design (spectral count)
You can define the experimental design of the analysis in the Quantitation Experimental Design page:
- Define the number of experimental biological states to compare
- Attribute a custom name to each State by clicking on "State n", edit the label. The attributed label will appear as tooltip in the results page
- You can also attribute custom names to the job items (these are the inter-job replicates names). The attributed label will appear as tooltip in the results page
- Attribute a state to a job by moving the cross vertically in each column
- Define the number of Ratios to be calculated and define Nominator and Denominator for each of them
- Click on the 'Submit' button (top panel). A progress bar is shown, and the result page appear in a new window. The calculation might take a few seconds to a few minutes, depending on the size of the identification results.
Evaluate the Results (isobaric labeling)
The main result page is divided into three sections:
- A horizontal bar with an export button. Three exports are available:
- A protein Ratios Summary in xml format,
- A Selected Protein Ratios in xml format (only the information about the selected protein is exported)
- An excel export
- A Protein Global Report table on the left side: list and ratio of quantified proteins
- The section on the right contains three tabs to visualise some more details:
- The Tables tab (fig. quanti-result-01) contains two tables:
- A Protein Details table on the right side: for a selected protein in the Protein Global Report, this table provides detailed information on each calculated protein ratio
- A Peptide Details table on the bottom-right side: for a selected protein in the Protein Global Report, this table provides detailed information at the peptide level. Click on a Score value to visualise the corresponding protein match details view; click on an Intensity value to open the corresponding peptide match details view
- The Protein Chart tab (fig. quanti-result-02) displays a protein ratios distribution graph. Each ratio distribution is represented in one color. The number of bins in the x axis (given in log(2) scale) can be changed using the vertical cursor
- The Peptide Chart tab (fig. quanti-result-03) opens a 2D scatter plot of the peptide intensity pairs for each chosen ratios, for a selected protein. Numerator and denominator intensities are represented in the Y and X axis, respectively. One color is attributed for each selected ratio.
- The Tables tab (fig. quanti-result-01) contains two tables:
The content of all columns in each table can be sorted by clicking on the column title. Ordered sorting of multiple columns can be done by keeping the CTRL key pushed while selecting the columns. The order of the columns can also be changed by drag-and-drop of the titles. The width of the columns can be adjusted. Tooltips are available for cut texts and to explicit numbered labels (states,replicates).
Fig. quanti-result-03 is representing a protein analysed in 3 states. In this graph, a ratio of 1/1 is seen as a cloud of dots centered on the diagonal; an overexpression is represented as a cloud of dots above the diagonal, an underexpression as a cloud of dots below the diagonal. The green dots and the blue squares represent similar overexpression of state 3 over state 1 (labeled as Ratio 3/1) and of state 3 over state 2 (Ratio 3/2), and the orange losanges represent a stable behavior between state 1 and 2.
Evaluate the Results (spectral count)
The main result page is constituted by three sections:
- A horizontal bar with an export button. Three exports are available:
- A protein Ratios Summary in xml format,
- A Selected Protein Ratios in xml format (only the information about the selected protein is exported)
- An excel export
- A Protein Global Report table on the left side: it provides the list and ratio(s) of quantified proteins
- A protein ratios distribution graph on the right-side. Each ratio distribution is represented in one color. The number of bins in the x axis (given in log(2) scale) can be changed using the vertical cursor
- A Protein Details table on the bottom: for a selected protein in the Protein Global Report, this table provides detailed information on each calculated protein ratio. Click on the Nominator and denominator emPAI values to open corresponding protein match details.
The content of all columns in each table can be sorted by clicking on the column title. Ordered sorting of multiple columns can be done by keeping the CTRL key pushed while selecting the columns. The order of the columns can also be changed by drag-and-drop of the titles. The width of the columns can be adjusted. Tooltips are available for cut texts and to explicit numbered labels (states,replicates).
