Proteomics software available in the public domain.
Pratik Jagtap Minnesota Supercomputing institute Two-Dimensional gel electrophoresis
pI Mw
Proteins are resolved based on their isolelectric point (using isoelectric focusing) and then molecular weight (using SDS-PAGE).
Gels are compared, differentially expressed proteins are excised and identified. Proteomics Fifteen Years Ago… Proteomics Fifteen Years Ago…
Mass Data Extrac on. Search Analysis So ware that correlates the Spectrometry algorithm protein ID to the excised gel spot. Two-Dimensional gel electrophoresis
pI Mw
2DGE : High molecular weight proteins, low molecular weight proteins, proteins with extreme isoelectric points, membrane proteins were underrepresented in the analysis. Multi-Dimensional Protein Identification Technology Proteomics workflow
Protein Peptide Fragmentation
Search against database. Mass spectrum mass spectrometry Mass Spectrometers & data formats
Thermofinnigan Sequest Xcalibur / .raw .dta .out Life Technologies ProteinPilot Analyst / .wiff ; .t2d .t2d .group
Waters Masslynx / .raw
Bruker X! tandem .baf .xml mzxml mzData OMSSA pepxml protxml .xml .omx Mascot mzml .mgf .dat Proteo-Informatics
Data Extrac on. Sta s cal valida on of Mass Data Conversion. Search Targeted pep de and Quan ta ve Spectrometry algorithm Proteomics protein Tools. De novo iden fica ons. Tools. Spectral Data Dissemina on Matching Data extraction Data Extrac on. Mass Spectrometry ReAdW http://www.ionsource.com/functional_reviews/readw/t2x_update_readw.htm ReAdW converts Xcalibur .raw files to universal mzXML format.
T2D Extractor https://www.prime-sdms.org/PRIMEInstallationSite/MSViewer/T2DExtractor.zip
• A tool that can access the Applied Biosystem’s MALDI-TOF/TOF 4700 and 4800 database and can extract T2D files as well as peak lists. • It can be used to extract individual spectra, runs, or entire spotsets. MS/MS peaklists are provided in .mgf formats. • Runs on Java 1.5 platform. • LCMS Peaklist Extractor – Batch mode tool for extracting concatenated .mgf peaklist files. • Quantitation Extractor – Batch mode tool for extracting areas for peaks in MS/MS spectra. Mass Spectrometers & data formats
Thermofinnigan Mascot Xcalibur / .raw .mgf .dat Life Technologies Sequest Analyst / .wiff ; .t2d .dta .out
Waters X! tandem Masslynx / .raw .xml
Bruker OMSSA .baf .xml .omx mzxml mzData pepxml protxml Data Conversion. mzml Mass Spectrometry data conversion Data Conversion. Mass Spectrometry mzXML2Other http://www.proteomecommons.org/current/522/ Converter from mzXML to sequest dta, mascot generic and micromass pkl formats.
Peak List Conversion Utility (Java Web Start) https://proteomecommons.org/tool.jsp?i=1012 The ProteomeCommons.org IO Framework's tool for converting peak list and spectrum files between different formats. The tool can also merge multiple peak lists into a single concatinated peak list. The tools uses Java Web Start and runs locally on your computer.
MassMatrix File Conversion Tools http://searcher.rrc.uic.edu/mm-docs/downloads /MM_File_Conversion_1p0.exe These tools convert between common input formats: .RAW, .mzXML, .MGF. search algorithm
Data Extrac on. Mass Data Conversion. Search Spectrometry algorithm SEARCH ALGORITHM Search algorithm X!tandem & the GPM Search algorithm
http://www.thegpm.org/TANDEM/index.html
• X! Tandem can be utilized as a web-based application or deployed locally using precompiled binaries and FASTA-formatted files. • X!Tandem takes inputs in .xml format and outputs .xml format. • The data analysis components consist of Input file ; FASTA, Taxonomy; Parameters and output. • Central Axiom : “For each identifiable protein, there is at least one detectable tryptic peptide.” • Extensively search for modified/ non-enzymatic peptides only on identified proteins. • How far is the top-scoring match from the rest of the pack? Uses E-value. Much faster than Sequest’s Xcorr.
The Global Proteome Machine Organization • X!Hunter • X! P3 • Common OMSSA Search algorithm
http://pubchem.ncbi.nlm.nih.gov/omssa/
• OMSSA takes experimental ms/ms spectra, filters noise peaks, extracts m/z values, and then compares these m/z values to calculated m/z values derived from peptides produced by an in silico digestion of a protein sequence library.
• Calculates E-value as a discriminant score. • An E-value for a hit is a score that is the expected number of random hits from a search library to a given spectrum such that the random hits have an equal or better score than the hit.
• It uses classical hypothesis testing based on type of statistical model that is used in BLAST.
• Faster; Runs on all platforms Search maxquant algorithm
http://www.maxquant.org/
• MaxQuant is an integrated suite of algorithms specifically developed for high- resolution, quantitative MS data.
• MaxQuant detects peaks, isotope clusters and stable amino acid isotope-labeled (SILAC) peptide pairs as three-dimensional objects in m/z, elution time and signal intensity space.
• By integrating multiple mass measurements, mass accuracy in the p.p.b. range is achieved.
• MaxQuant quantifies several hundred thousand peptides per SILAC-proteome experiment. De novo tools
Data Extrac on. Mass Data Conversion. Search Spectrometry algorithm De novo Tools. De novo de novo analysis Tools.
Protein Peptide Fragmentation
Mass spectrum Search against database.
• De novo Analysis : Generate sequence from spectrum and match against database by using BLAST pepnovo De novo Tools. h p://pep de.ucsd.edu/pepnovo.html
• PepNovo is a software for de novo sequencing of peptides from mass spectra.
• PepNovo uses a probabilistic network to model the peptide fragmentation events in a mass spectrometer.
• In addition, it uses a likelihood ratio hypothesis test to determine if the peaks observed in the mass spectrum are more likely to have been produced under the fragmentation model, than under a probabilistic model that treats the appearance of peaks as random events. De novo lutefisk Tools. http://sourceforge.net/projects/lutefiskxp
• LUTEFISK uses a graph theory approach for de novo peptide sequence determinations from low-energy collision-induced dissociation (CID) data of tryptic peptides.
• Lutefisk converts all of the ions into their corresponding b-ion masses by making N- and C-terminal “evidence lists” that contain evidence for cleavage at every possible b-ion mass. Once the sequence spectrum has been established, the program proceeds by tracing sequences starting at the N-terminus.
• Highest ranked sequences are subjected to a cross-correlation analysis and scores are combined and normalized to produce a final score and ranking. spectral matching
Data Extrac on. Mass Data Conversion. Search Spectrometry algorithm De novo Tools. Spectral Matching Spectral x!hunter Matching http://www.thegpm.org
• X! Hunter is a search engine that compares experimentally observed spectra directly with a library of spectra that have been confidently assigned to a particular peptide sequence (an Annotated Spectrum Library, or ASL).
• It can identify proteins using information from large number of spectra in GPMDB database.
• Creation of ASLs : 1) Confident assignments for human and yeast peptides were extracted from GPMDB. 2) Replicate observations of the same peptide were averaged together and a final list of averaged peptide spectra was produced.
• Because the sequence modifications and cleavage sites for the peptides in the sequence library are already known, it is not necessary to specify as many parameters for this type of search as in more conventional search engines.
• This type of pattern matching tool is ideal for applications such as biomarker discovery. Spectral MS-Clustering Matching http://proteomics.bioprojects.org/MassSpec
MS-Clustering of MS/MS spectra takes advantage of dataset redundancy by identifying multiple spectra of the same peptide and replacing them with a single representative spectrum.
Analyzing only representative spectra results in significant speed-up of MS/MS database searches.
Large MS/MS data sets (over 10 million spectra) were reduced to smaller datasets and resulted in higher number of peptide identifications as compared to regular nonclustered searches. Data Extrac on. Sta s cal valida on of Mass Data Conversion. Search pep de and Spectrometry algorithm protein De novo iden fica ons. Tools. Spectral Matching Sta s cal valida on of pep de and protein iden fica ons. Trans-proteomic pipeline
Sta s cal valida on of pep de and protein iden fica ons.
Trans-Proteomic Pipeline (TPP) is a data analysis pipeline for the analysis of LC/ MS/MS proteomics data. TPP includes modules for validation of database search results, quantitation of isotopically labeled samples, and validation of protein identifications, as well as tools for viewing raw LC/MS data, peptide identification results, and protein identification results. The XML backbone of this pipeline enables a uniform analysis for LC/MS/MS data generated by a wide variety of mass spectrometer types, and assigned peptides using a wide variety of database search engines. peparml
Sta s cal valida on of pep de and http://mac.softpedia.com/get/Math-Scientific/PepArML.shtml protein iden fica ons. Feature extraction X!Tandem PepArML Mascot
OMSSA
Other
A model-free, result-combining peptide identification arbiter via machine learning. Quantitative tools
Data Extrac on. Sta s cal valida on of Mass Data Conversion. Search pep de and Quan ta ve Spectrometry algorithm protein Tools. De novo iden fica ons. Tools. Spectral Matching iTRAQ™ : Isobaric Tags for Relative and Absolute Quantification.
Isobaric Tag (Total mass = 145)
Peptide Reporter Balance Reactive Charged Neutral loss Group
Trypsin digest 114 31 PRG +
114 115 30 PRG + Mix 115 -N H MS -N 116 H -N MS/MS H -N H 117 116 29 PRG + MS 100 100 90 80 [Reporter-Balance-Peptide] 90 70 60 50 80 40% Intensity 30 70 20 10 60 0 50 Mass (m/z) QGQPIGLGEASNDTWI
% Intensity 40 117 28 PRG + 30 TTK 20 10 072.0 509.8 947.6 1385.4 1823.2 2261.0 Mass (m/z) Proteomics Quantitatition i-tracker Quan ta ve Tools. http://www.dasi.org.uk/download/itracker.htm
• i-Tracker is an open-source peptide quantitation algorithm that allows the user to extract reporter ion peak ratios from non-centroided peak lists.
• The algorithm uses .dta and .mgf files as inputs. The reporter ion areas are calculated and corrected for their purity.
• The .csv output of i-Tracker allows for the relative comparison of the iTRAQ labeled peptides. TPP Quantitative Tools ASAP ratio, Xpress and libra Quan ta ve Tools. ASAPRatio http://tools.proteomecenter.org/ASAPRatio.php
Automated Statistical Analysis on Protein Ratio (ASAPRatio) accurately calculates the relative abundances of proteins and the corresponding confidence intervals from ICAT- type ESI-LC/MS data. XPRESS http://tools.proteomecenter.org/XPRESS.php
The XPRESS software calculates the relative abundance of proteins, such as those obtained from an ICAT-reagent labeled experiment, by reconstructing the light and heavy elution profiles of the precursor ions and determining the elution areas of each peak.
LIBRA http://tools.proteomecenter.org/wiki/index.php?title=Software:Libra
Libra is a module within the trans-proteomic pipeline to perform quantification on MS/MS spectra that have iTRAQ labeled samples. APEX
Quan ta ve http://pfgrc.jcvi.org/index.php/bioinformatics/ Tools.
• The APEX Quantitative Proteomics Tool is a free and open source Java implementation of the APEX technique for the absolute quantitation of proteins based on standard LC- MS/MS proteomics data.
• It uses machine learning techniques to improve quantitation accuracy for label- free technique.
• The APEX Tool provides an intuitive user interface, an integrated help system, and rich documentation.
• A tutorial and sample data set is included to help first time users become acquainted with the system. maxquant Quan ta ve Tool http://www.maxquant.org/
• MaxQuant quantifies several hundred thousand peptides per SILAC-proteome experiment. Targeted Proteomics
Data Extrac on. Sta s cal valida on of Mass Data Conversion. Search Targeted pep de and Quan ta ve Spectrometry algorithm Proteomics protein Tools. De novo iden fica ons. Tools. Spectral Matching Targeted Proteomics Biochemistry vs Proteomics
Targeted proteomics vs Shotgun Proteomics MRM Targeted Proteomics
Selectivity, Sensitivity and Dynamic Range…
Quantitative Proteomics Results Prediction
Choose and Optimize Transistions TIQAM Targeted Proteomics http://tools.proteomecenter.org/TIQAM/TIQAM.html
• TIQAM generates MRM transition lists and identifies the best performing transitions from MRM pre-experiments.
• In addition TIQAM provides a viewer to validate transitions by MRM-triggered MS/MS experiments.
• All the peptide and transition information is stored in a database to enable smart retrieval of the validated transitions for quantitative analysis.
• Commercial softwares : MRMPilot (Applied Biosystems), SRM Workflow Software (Thermo Scientific), VerifyE (Waters) and Optimizer (Agilent Technologies). X! P3 Targeted Proteomics http://www.thegpm.org
ftp://ftp.thegpm.org/proteotypic_peptide_profiles
• Uses identification of “proteotypic peptides” for identification of a protein. Because there will only be a few proteotypic peptides for a protein, it improves both the speed and accuracy of the resultant protein identifications.
• The X! P3 (Proteotypic Peptide Profiler) project uses the following steps :
1. In the first round, the spectrum data set is examined for the presence of proteotypic peptides. This is done by querying GPMDB to find the best peptides representative of a particular protein. 2. The full protein sequences of the proteins identified in the first round are then pulled from a sequence library. 3. Using this small set of full sequences, multiple rounds of refinement are performed to extract all of the non-proteotypic peptides from the full spectrum data set
• An X! P3 server has been established for two model organisms, namely Homo sapiens and Saccharomyces cerevisiae, as well as several commonly observed experimental artifacts, such as BSA and trypsin. Data Extrac on. Sta s cal valida on of Mass Data Conversion. Search Targeted pep de and Quan ta ve Spectrometry algorithm Proteomics protein Tools. De novo iden fica ons. Tools. Spectral Data Dissemina on Matching
Your Answer is going to be determined by the ques on asked. Data Dissemina on Prestomic
http://code.google.com/p/prestomic
• An open-source suite of tools for storing data and for presenting the data in a user-friendly format via a browser.
• The program was developed using mostly Perl. Data Dissemina on Tranche
https://proteomecommons.org/tranche/
Tranche is a free and open source file sharing tool that enables the storage of large amounts of data. Designed and built with scientists and researchers in mind, Tranche can handle very large data sets, is secure, is scalable, and all data sets are citable in scientific journals. Proteomic pipelines that use Open-source software. CPAS http://proteomics.fhrc.org/CPAS Open source toolkit that integrates open source proteomics tools along with existing commercial software.
CORRA http://tools.proteomecenter.org/Corra/corra.html Statistical Analysis tools for Quantitative proteomics
SysPIMP http://pimp.starflr.info Identify mutated proteins from mass spectrometry results.
SwissPIT http://swisspit.cscs.ch Multitool platform that promotes use of multiple search algorithms. mMass data miner http://mmass.biographics.cz
The OpenMS Proteomics Pipeline http://ww.openms.de Protip
Raw Data from mzxml format Mgf Orbitrap format
dta format
X!TANDEM OMSSA SEQUEST MASCOT search search search search
Scaffold Analysis
Scaffold Viewer performing multiple searches through Protip
HUMAN DATASET
8162 8400 7443 Mgf mzxml format 6962 7200 6554 format
6000 5522 5486 5137 4800 dta format 3600 # of peptides 2400 X!TANDEM OMSSA SEQUEST MASCOT search search search search 1200 491 462 441 441 411 401 370
0 # of proteins Scaffold Mascot Sequest Analysis X! tandem All Together Sequest + Mascot X! tandem + Mascot Sequest + X! tandem LAST WORD…
Questions ? Pratik Jagtap [email protected]