ProteoSign is an online service for protein differential expression (or abundance) analysis designed with the end-proteomics user in mind.
Borrowing from the experience of the microarray field, ProteoSign software utilizes the well-established Linear Models For Microarray Data (LIMMA) methodology for assessing statistical significance in protein abundance differences between two or more proteome states.
ProteoSign aims to fully automate the process of statistically evaluating differential expression in mass spectrometry-based bottom-up (shotgun) quantitative proteomics data by requiring minimal user interaction time and generating publication-quality data plots.
Click Start to begin your analysis.
New features added to ProteoSign ver. 2.0! Click here to see what's new!
The License of Protesign is BSD Click here to see it.
Step 1
Please read the text below. When you are ready, upload your dataset and click Next. Alternatively, you can choose from available test datasets.
The peptide spectrum match (PSM) file, comprising the necessary information required for protein quantitation, has to be produced manually through the PD desktop application (see the Export→To Text option under the File menu).
The PSM file must be created from within a PD report which combines the information from all relevant MS analyses (i.e. replicates, conditions etc). Such a PD report is sometimes required to be created based on separate reports and is referred to as a multi-consensus report.
Before exporting the information, please disable peptide grouping and edit the report's Quantification Method by configuring the Ratio Calculation parameters as shown below. The following screen-shots show where the aforementioned changes can be made from within the PD application.
Disabling peptide grouping.Editing ratio calculation parameters.
— MaxQuant (MQ) v1.3.0.5+ 'proteinGroups' and 'evidence' files.
The input file(s) should be made out of merging multiple LC-MS/MS runs (fractionation, replication) of a single, possibly multiplexed, experiment and should not be the result of merging different or parallel experiments. Thus, multi-experiment analysis is not supported. In addition, only single factor (one-way) analysis is currently supported.
Upload file(s)
Step 2
Please define the experimental design by assigning the appropriate structure coordinate to each file.
A coordinate specifies the biological replicate, technical replicate and fraction a raw file belongs to. Select one or more files from the table on the left, type the biological and technical replicate numbers they belong to in the boxes on the right, and click the Assign button. If multiple files are fractions of the same replicate, simply assign the same biological and technical replicate number to them, the order of the fractions does not matter. Use Shift to select a range of items. Right click on the table for extended functionality.
Please use the "Assign Condition" option in the right click menu to tell ProteoSign which Raw Files correspond to which condition.
Experimental structure coordinate
You have chosen to use Replication Multiplexing. You can Undo this or Review your Replication Multiplexing options.
Load parameters...
Step 3
Define the experimental parameters and click Submit.
— Raw data were quantified with Proteome Discoverer™
— Experiment ID
— Experiment description
— Conditions to compare
Show advanced parameters
— Protein (as opposed to peptide) quantitation?
— Non-labelled "background" species present?
— "Background" species
— Quantitation filtering?
?For labelled experiments only. If enabled, two options will become available: a) singlets (peptides with no signal at the MS level in all but one labelled version) will be excluded from protein quantitation (this is referred to as peptide-level filtering). b) Proteins identified just by peptides with a certain label will be excluded from the analysis (this is referred to as protein-level filtering).
— Filtering based on which label?
?The singlet label.
— Type of filtering: Peptide-level (as opposed to protein-level)?
— Label Swap options...
— Miscellaneous...
— Expression enrichment analysis
— Target organism:
Save/Load parameters...
Step 4
Save parameters...
End
Please download your results. Click Reset to start again. Thank you for using ProteoSign.
Functional enrichment analysis:?ProteoSign runs a functional enrichment analysis for all differentially expressed proteins to give more insight into the respective cellular procedures. One analysis is run for each pair of conditions compared. Choose the desirable condition pair from the list below and double click on a term to see more details.
Condition pair:
Feedback
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Select one or more (by holding the Ctrl key).
Select a dataset and press OK. You can also download it to your machine.
Select a condition to assign to the selected raw files or type a New condition in the box below.
ProteoSign says:
Please type the Name of the New condition:
Label Swap Assignment
Please select the Biological and Technical replicate where labels were swapped
Raw files affected:
Then, select the Labels that were swapped and click Add to assign the swap
Current label swaps assigned:
Change the default data analysis parameters used by ProteoSign and click OK
Proteins that were not quantified with at least different peptides in at least biological replicates will be disqualified.
Adjusted p-value threshold for differentially expressed proteins:
Thank you for using ProteoSign! Please leave us your feedback below:
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Replication Multiplexing
ProteoSign supports data sets that have biological or technical replicates represented as different tags. Please choose the options below that best describe your experiment and click Next: If you have no technical replicates select "Raw Files" as the corresponding option
My biological replicates are represented as different
Raw Files
Tags
My technical replicates are represented as different
Raw Files
Tags
My conditions are represented as different
Raw Files
Tags
Please assign the corresponding elements to your Raw files the fractions will be updated automatically
The peptide spectrum match (PSM) file, comprising the necessary information required for protein quantitation, has to be produced manually through the PD desktop application (see the Export→To Text option under the File menu).
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