Proteomics software at MSI. Pratik Jagtap Minnesota Supercomputing institute http://www.mass.msi.umn.edu/
Proteomics software at MSI. proteomics : emerging technology proteomics workflow search algorithms de novo analysis : Peaks Statistical validation of protein identification Quantitative tools Targeted proteomics Data dissemination : Tranche
Proteomics Fifteen Years Ago
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 Mass Spectrometry Data Extrac5on. Search algorithm Analysis So9ware that correlates the protein ID to the excised gel spot.
Genomics Genomics : Number of Databases for search and Shotgun analysis.
Multi-Dimensional Protein Identification Technology
mass spectrometry
Mass Spectrometers & data formats Thermofinnigan Xcalibur /.raw Life Technologies Analyst /.wiff ;.t2d Waters Masslynx /.raw Bruker.baf Sequest ProteinPilot X! tandem OMSSA Mascot
Proteomics workflow Protein Peptide Fragmentation Mass spectrum Search against database.
mass spectrum Intensity Mass / charge
Proteo-Informatics Mass Spectrometry Mass spectral data. Search algorithm De novo Tools. Sta5s5cal valida5on of pep5de and protein iden5fica5ons. Quan5ta5ve Tools. Targeted Proteomics Data Dissemina5on
search algorithm Mass Spectrometry Mass spectral data. Search algorithm
search algorithm Mass Spectrometry Mass spectral data. Search algorithm Sequest X!tandem OMSSA MaxQuant RDC : sdvlapp32 ProteinPilot CPC9 ; CGL 138. Mascot https://sequest7.msi.umn.edu/mascot
Protip / TINT Raw Data from Orbitrap mzxml format Mgf format dta format X!TANDEM search OMSSA search SEQUEST search MASCOT search Scaffold Analysis Scaffold Viewer Powered by
performing multiple searches through Protip HUMAN DATASET 8400 7200 8162 6554 6962 7443 6000 5522 5137 5486 4800 3600 2400 1200 0 401 370 411 491 441 MASCOT search Sequest X! tandem 441 Mascot All Together # of 462 proteins Sequest + Mascot Sequest + X! tandem X! tandem + Mascot Powered by
Search algorithm PROTEINPILOT
ProteinPilot accounts for more spectra by screening for large number of modifica@ons. Glu >pyro Glu itraq4plex Methylthio No itraq4plex Amino(Y) Arg >GluSA(R) Ca@on:Cu[I](D) Ca@on:Cu[I](E) Ca@on:K(D) Ca@on:Na(D) Ca@on:Na(E) Deamidated(N) Deamidated(Q) Dehydrated(D) Dehydrated(E) Dehydrated(S) Dehydrated(T) Delta:H(4)C(2)(H) Dethiomethyl(M) Dioxida@on(M) Dioxida@on(W) itraq4plex(h) itraq4plex(k) itraq4plex(s) itraq4plex(t) itraq4plex(y) Methylthio(C) G A substitution Hydroxy-proline Dioxidation (W) itraq4plex(ser)
Search algorithm maxquant 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 isotopelabeled (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 SILACproteome experiment.
De novo Tools. PEAKS Options No DB de novo Sequence but no ID Protein DB Multiple software Homologous DB Protein ID inchorus SPIDER Sequence with ID high confidence Sequence ID Sequence with putative ID data in different formats Protein ID PTM Finder PTMs Protein ID PEAKS Q quantity
De novo Tools. PEAKS resources at MSI PEAKS Online http://sequest5.msi.umn.edu:8080/peaksonline/ Get a password from Tu. Set up a search using peaklist. Monitor your search status. Links for.anz files that can be used further in PEAKS Client. PEAKS Client v4.5 PEAKS Client available at CGL : CPC7 and CPC10. Use remote access to cpc7.msi.umn.edu or cpc10.msi.umn.edu Use your.anz file (generated from Online search) for further analysis.
Sta5s5cal valida5on of pep5de and protein iden5fica5ons.
Scaffold Sta5s5cal valida5on of pep5de and protein iden5fica5ons. https://www.msi.umn.edu/sw/scaffold-for-pro
Proteomics Quantitatition
itraq : Isobaric Tags for Relative and Absolute Quantification. Isobaric Tag (Total mass = 145) Reporter Charged Balance Neutral loss Peptide Reactive Group Trypsin digest 114 31 PRG + 115 30 PRG 116 29 PRG 117 28 PRG + + + Mix MS -N H -N H -N H -N H MS [Reporter-Balance-Peptide] MS/MS % Intensity 100 90 80 70 60 50 40 114 115 116 117 % Intensity 100 90 80 70 60 50 40 30 20 10 0 Mass (m/z) QGQPIGLGEASNDTWI TTK 30 20 10 0 72.0 509.8 947.6 1385.4 1823.2 2261.0 Mass (m/z)
Quan5ta5ve Tool maxquant http://www.maxquant.org/ MaxQuant quantifies several hundred thousand peptides per SILAC-proteome experiment.
ProteinPilot supports normaliza@on and quan@fica@on of itraq datasets.
Targeted Proteomics MRM Selectivity, Sensitivity and Dynamic Range Quantitative Proteomics Results Prediction Choose and Optimize Transistions
Data Dissemina5on 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.
LAST WORD Sequest X! tandem OMSSA MaxQuant ProteinPilot Mascot Scaffold PEAKS TPP Tranche Trans-proteomic Pipeline Pipeline ProTIP http://sitemaker.umich.edu/iwsmoi Questions? Pratik Jagtap pratik@msi.umn.edu http://twitter.com/pratikomics