We focus on the automation of proteomics data analysis and the development of processing algorithms and workflows. Our research interests are
Multiplexed MRM/PRM experiments with 100s of peptides involves integration of information on the target peptides from multiple sources to plan the experiment, and advanced algorithms are required for processing the acquired data.
MADpipe is a software suite for MRM-assay design that includes:
1) PeptideTracker, a knowledge base, containing empirical information on light and heavy labeled peptides in human and mouse
2) PeptidePicker Library, a pre-compiled library of all possible peptide surrogates for all human proteins [PMID: 26232110]
3) PeptidePicker for selecting proteotypic peptides, based on information about the protein and the suitability of its tryptic peptides for MRM from six on-line knowledge bases [PMID: 24769191]
4) Qualis-SIS, for rapid post-analytical calculation and visualization of MRM data [PMID: 25546269].
All tools use simple data formats allowing easy manipulation and use with each other.
Link to MADpipe Web interface
Selecting the most appropriate surrogate peptides to represent
a target protein is a major component of experimental design in
Multiple Reaction Monitoring (MRM). Our software PeptidePicker with its
v-score remains distinctive in its approach of integrating information
about the proteins, their tryptic peptides, and the suitability of
these peptides for MRM that is available online. PeptidePicker Library
is a compiled a list of all possible surrogate peptides of the human
proteome. Using our stringent selection criteria, the list includes
165k suitable MRM peptides covering 17k proteins of the human reviewed
proteins in UniProtKB. Compared to average of 2-4min per protein for
retrieving and integrating the information, the precompiled list
includes all peptides available instantly. This allows a more cohesive
and faster design of a multiplexed MRM experiment and provides insights
into evidence for a protein's existence. The Library is avilable for
download as well as for direct online search using our MADpipe software.
Link to PeptidePciker libray publication
Qualis-SISMultiplexed targeted quantitative proteomics typically utilizes multiple reaction monitoring and allows the optimized quantification of a large number of proteins. One challenge, however, is the large amount of data that needs to be reviewed, analyzed, and interpreted. Different vendors provide software for their instruments, which determine the recorded responses of the heavy and endogenous peptides and perform the response-curve integration. Bringing multiplexed data together and generating standard curves is often an off-line step accomplished, for example, with spreadsheet software. This can be laborious, as it requires determining the concentration levels that meet the required accuracy and precision criteria in an iterative process. We present here a computer program, Qualis-SIS, that generates standard curves from multiplexed MRM experiments and determines analyte concentrations in biological samples. Multiple level-removal algorithms and acceptance criteria for concentration levels are implemented. When used to apply the standard curve to new samples, the software flags each measurement according to its quality. From the user's perspective, the data processing is instantaneous due to the reactivity paradigm used, and the user can download the results of the stepwise calculations for further processing, if necessary. This allows for more consistent data analysis and can dramatically accelerate the downstream data analysis.
One challenge in Multiple Reaction Monitoring Proteomics analysis is identifying the most appropriate surrogate peptides to represent target proteins. These peptides are synthesized as stable isotope standards (SIS), incorporating 15N/13C isotope labeled C-terminal Arginine or Lysine to enable the differentiation between endogenous and surrogate by mass spectrometry. Spiking in a known amount of SIS during sample processing allows for the generation of endogenous to SIS ratios and thus accurate quantitation. These surrogate peptides should anyhow be efficiently liberated during trypsin digestion, free of post translational modifications and variants and unique within the target proteome. PeptidePicker is a scientific workflow to automatically generate the most appropriate surrogate peptides to represent target proteins in an LC-MRM/MS analysis. It integrates the information available online in UniProtKB, ExPASy, PeptideAtlas, and GPM. The scientific workflow allows linking own database to test whether already used peptides are indeed the most appropriate one or not. The modularity of the developed tool makes it ready for extensions and incorporating more selection criteria. PeptidePicker has also a Web interface where the researcher provides only the protein accession number and the organism. Currently the tool works for human and mouse.
PeptideTrackerThe use of stable-isotope labeled (SIS) peptides in MRM experiments is a key factor contributing to high-accuracy quantitative measurements. MRM experiments are also amenable to high degrees of multiplexing, allowing hundreds of peptides to be quantitated in a single LC/MS run. Laboratories who routinely perform such analyses can therefore quickly amass inventories of hundreds to thousands of SIS peptides, which can have hundreds of metadata points associated with each one. PeptideTracker is software for tracking all of the information associated with each SIS peptides within a laboratory.
The software is based on a relational database with a Web interface. Hierarchical permission schemes permit access restrictions based upon assigned roles. The structure of the software tool was built to reflect the actual workflow of peptides as they move though the laboratory from synthesis to optimization on MS instruments to the creation of SIS peptide mixtures. Data and metadata pertaining to the SIS peptides, mixtures and the corresponding experiments has been written so that reports can be queried and formatted for output in formats that are best suited to the function of particular laboratory members. The software is not only a repository to store all information about SIS peptides; it also allows exporting the optimization parameters of each peptide so they can be used for designing MRM assays.
General Mass Spectrometry-based Proteomics Data Analysis
The UVic-GBC Proteomics Centre houses also 3rd party proteomics
software that cover most aspects of MS/MS datasets analysis and
interpretation. The folowing is a list of some of these software:
Mascot, PEAKS, X!Tandem, SEQUEST, Trans Proteomics Pipeline, Scaffold,
Thermo Fisher Scientfic Proteome Discoverer, Agilent MassHunter,Thermo
Fisher Scientific Xcalibur, Bruker Data Analysis.