Automated peptide mapping and protein-topographical annotation of proteomics data
- Pavankumar Videm†1, 2,
- Deepika Gunasekaran†1,
- Bernd Schröder3,
- Bettina Mayer1,
- Martin L Biniossek1 and
- Oliver Schilling1, 4, 5Email author
© Videm et al.; licensee BioMed Central Ltd. 2014
Received: 6 March 2014
Accepted: 18 June 2014
Published: 19 June 2014
In quantitative proteomics, peptide mapping is a valuable approach to combine positional quantitative information with topographical and domain information of proteins. Quantitative proteomic analysis of cell surface shedding is an exemplary application area of this approach.
We developed ImproViser (http://www.improviser.uni-freiburg.de) for fully automated peptide mapping of quantitative proteomics data in the protXML data. The tool generates sortable and graphically annotated output, which can be easily shared with further users. As an exemplary application, we show its usage in the proteomic analysis of regulated intramembrane proteolysis.
ImproViser is the first tool to enable automated peptide mapping of the widely-used protXML format.
Peptide mapping is increasingly recognized as a valuable tool in quantitative proteomics. It integrates quantitative information of individual, typically tryptic, peptides with topographical protein annotation such as individual domains. Manual peptide mapping has established that matrix metalloprotease (MMP)-2 proteolytically releases the chemokine fractalkine into the pericellular milieu . Peptide mapping is also crucial for correct functional annotation, e.g. distinguishing collagen cleavage products with signaling function from the actual collagen protein with a predominantly structural role .
Signal-peptide-peptidase-like (SPPL) proteases SPPL2a and –b cleave transmembrane proteins within the lipid bilayer with a preference for transmembrane proteins in type 2 orientation . The few annotated substrates of SPPL2a and -b include tumor necrosis factor [4, 5], the Fas ligand  and the invariant chain (CD74) of the major histocompatibility class II complex [7–10]. Common features of SPPL2a/b substrates include a short cytoplasmic tail and a large ectodomain. SPPL proteases release the cytoplasmic tail after initial shedding of the ectodomain by other proteases. From a proteomic perspective, quantitative alterations of the cytoplasmic tail are typically overshadowed by peptides stemming from the ectodomain. This makes peptide mapping useful in the proteomic analysis of SPPL proteolysis.
Some proteomic applications already include peptide mapping. A strategy termed PROTOMAP combines high coverage peptide mapping with size shift analysis to detect proteolytic truncation of proteins . A novel tool termed QARIP  works with the proteomic software Maxquant  to analyze cell surface shedding by automated peptide mapping. Similarly, the PROTTER tool integrates experimental proteomics data with protein sequence annotations .
protXML is a well-established format to report protein identification and quantitation based on liquid chromatography–tandem mass spectrometry (LC–MS/MS). protXML is most prominently implemented by the Trans Proteomic Pipeline (TPP) , a set of open–source tools for quantitative proteomic data analysis. A large user community extensively employs the TPP which is known for supporting a large range of data formats and mass spectrometers .
Deconvolute global protein ratios by spatially resolving the underlying peptide ratios.
Facilitate the analysis of cell surface shedding by distinguishing extra- and intracellular ratios for membrane–spanning proteins.
Facilitate interpretation of proteomic results by linking protein IDs to the corresponding UniProt information .
Share these results with non–expert users and collaborators in a straightforward manner.
To fulfill these requirements, we developed ImproViser (Improved Visualizer of protXML data), which is freely accessible at http://www.improviser.uni-freiburg.de.
ImproViser supports protein identifications originating from sequence databases that adhere to UniProt  or International Protein Index (IPI) nomenclature . The tool obtains UniProt identifiers from the protXML file and subsequently retrieves the following entry-specific information from the UniProt database: recommended name; molecular weight; length; topological information such as presence and location of transmembrane regions, N-terminal signal peptides, cytoplasmic, and extra-cytoplasmic (e.g. extracellular, and luminal) domains.
Invert H and L
This function enables the user to invert the light to heavy ratios in the ProtXML file to heavy to light ratios.
Validate ASAPratio with Xpress
Elaborate list of peptides
Selecting this function displays the list of all occurrences of a peptide (in case they are identified more than once, by default the tool chooses the peptide with highest Peptide Prophet probability score).
This function enables the user to set the cutoff for the ProteinProphet probability score. Any protein with a score less than this cutoff is discarded.
Minimum peptide ratio
This function enables the user to set the minimum value allowed for light:heavy ratio of the peptide. This measure is then used for scaling of the peptide ratios.
Maximum peptide ratio
This function enables the user to set the maximum value allowed for light:heavy ratio of the peptide. This measure is then used for scaling of the peptide ratios.
Negative no change zone
This function enables the user to set the negative threshold for light to heavy ratio of the peptide. i.e. the peptide ratios between the Zn and Zp thresholds are categorized together.
Positive no change zone
This function enables the user to set the positive threshold for light to heavy ratio of the peptide. i.e. the peptide ratios between the Zn and Zp thresholds are categorized together.
Protein entries are considered as being “valid” if they pass the criteria described above. For each valid protein entry, ImproViser retrieves annotation from UniProt as described above. In addition, the tool extracts the individual peptide L:H ratios (as determined by ASAPRatio) for each valid protein entry. Peptide ratios are normalized as described above, log2 transformed, and graphically mapped on the linear protein sequence using a red - green scale to visualize individual peptide ratios. For protein regions that are explicitly annotated as being cytoplasmic or extra-cytoplasmic, ImproViser calculates a novel average ratio.
ImproViser is accessible via http://www.improviser.uni-freiburg.de. A test data set is also available for download. The user uploads an input protXML file and the tool generates an output HTML file (named index.html), which enables a tabulated visualization of the input. The tool outlines the details of the identified proteins and peptides. It also enables the user to select proteins based on specific features such as presence of N-terminal signal peptides and presence of transmembrane regions. The tool further generates (a) a log file which contains a list of proteins that were discarded (named run_stats.out), (b) a .txt file containing the information about the average molecular weight of the proteins listed in the output HTML file (named average_molecular_weight.txt), (c) a .txt file describing the system requirements and browser compatibility for viewing the output HTML file in its intended format (named suppoted_browsers_and_os.txt), (d) folders for storing images which are displayed in the index.html file (named images, small_images), and (e) a folder for storing HTML file link for specific proteins (named index_files). ImproViser also copies the necessary java scripts and css files required for the script to generate the formatted output. The formatted output produced by the tool is supported by all css3 compatible web browsers. The above-mentioned files are compressed in a zip format and presented for download. In our experience, the file size is often below 10 MB, thus allowing for easy sharing with collaborators via e-mail or file transfer services.
Results and discussion
Application to proteomic analysis of SPPL - mediated intramembrane proteolysis
As outlined above, SPPL2a and SPPL2b typically cleave type 2 transmembrane proteins with short cytoplasmic tails following the initial proteolytic shedding of a larger ectodomain. For proteomic analysis of putative SPPL2a/b substrates, bone marrow derived dendritic cells (BMDCs) were prepared from mice deficient for both SPPL2a and SPPL2b (SPPL2a -/- SPPL2b -/- ). Control BMDCs were generated from bone marrow of wild-type mice. BMDC isolation and culture has been performed as described previously . Subsequently, cells were harvested and mechanically disrupted. Total cellular membranes were recovered by ultracentrifugation from a post-nuclear supernatant and washed with 100 mM sodium carbonate, pH 11.5, in order to enrich integral membrane proteins as described previously . Following tryptic digestion in the presence of the acid labile surfactant RapiGest (Waters), peptides were dimethylated with stable isotopic forms of formaldehyde as described previously [2, 22]. LC-MS/MS and corresponding data analysis with the TPP were also performed as described previously [2, 22]. The resulting prot.xml file was further analyzed by ImproViser.
Proteomic analysis of the murine BMDC membrane fraction
Total proteins identified and quantified
– with annotated transmembrane domain
– with annotated signal peptide sequence and with signal peptide sequence
– with annotated transmembrane domain but without signal peptide sequence
– with annotated transmembrane domain and quantified peptides of cytoplasmic localization
– with annotated transmembrane domain and quantified peptides of extra-cytoplasmic localization
It is an intrinsic feature of every peptide mapping approach that quantitations of protein domains are based on less peptide features than those for the entire protein. For example, the cytoplasmic tail of the invariant chain (CD74) of the major histocompatibility class II complex encompasses 29 amino acids with one tryptic peptide of 17 residues. The reduced number of peptide features employed in domain quantitation necessitates particular care in the interpretation of such results since individual peptide quantitations are prone to poor chromatographic resolution  or non-dynamic behaviour in quantitative proteomic analysis .
Peptide mapping is a useful additional level of proteomic data analysis. The ImproViser tool serves as a platform to automate this process and provides a graphical representation of protXML data, as highlighted by an exemplary proteomic analysis of regulated intramembrane proteolysis. We consider quantitative proteomic analysis of cell surface shedding to be a major application area of ImproViser. It might also be of interest for the proteomic analysis of other post-translational modifications such as phosphorylation.
Availability and requirements
Project name: ImproViser
Project home page: http://www.improviser.uni-freiburg.de
Operating system: Platform independent
Programming language: Perl
Other requirements: Requires web browsers that support css3, hence recent versions of Firefox, Chrome and Opera are recommended.
License: ImproViser is available freely online at http://www.improviser.uni-freiburg.de
Any restrictions to use by non-academics: none
The authors thank Sebastian Held and Franz Jehle for excellent technical assistance. O.S. is supported by grants of the Deutsche Forschungsgemeinschaft (DFG) (SCHI 871/2 and SCHI 871/5) and the SFB850, a starting grant of the European Research Council (Programme “Ideas” - Call identifier: ERC-2011-StG 282111-ProteaSys), and the Excellence Initiative of the German Federal and State Governments (EXC 294, BIOSS). B.S. is supported by the Deutsche Forschungsgemeinschaft as part of the SFB 877 and the Centre of Excellence “Inflammation at Interfaces”. The article processing charge was funded by the German Research Foundation (DFG) and the Albert Ludwigs University Freiburg in the funding program Open Access Publishing.
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