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…

Search algorithm 

   Mass Spectrometry 

Data Extrac5on.  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

Mascot

Sequest

X! tandem OMSSA

ProteinPilot

Proteomics workflow

Protein Peptide

Mass spectrum

Fragmentation

Search against database.

mass spectrum In

tens

ity

Mass / charge

Proteo-Informatics

   Mass Spectrometry 

Mass spectral data.  Search 

algorithm 

Sta5s5cal valida5on of pep5de and protein iden5fica5ons. De novo  

   Tools. 

  Data Dissemina5on 

Quan5ta5ve  Tools. 

Targeted  Proteomics 

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

dta format

X!TANDEM search

Scaffold Analysis

Scaffold Viewer

MASCOT search

SEQUEST search

Mgf format

OMSSA search

Powered by

performing multiple searches through Protip

MASCOT search

# of

pro

tein

s

5522 5137 

5486 

8162 

6554 6962 

7443 

401 

370 

411 

491 

441 

441 

462 

0 

1200 

2400 

3600 

4800 

6000 

7200 

8400 

Sequ

est 

X! ta

ndem

 

Mascot 

All Together 

Sequ

est +

 Mascot 

Sequ

est +

 X! tande

m 

X! ta

ndem

 + M

ascot 

HUMAN DATASET

Powered by

PROTEINPILOT Search algorithm 

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) 

Hydroxy-proline

G A substitution

iTRAQ4plex(Ser)

Dioxidation (W)

maxquant Search algorithm 

•  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.

http://www.maxquant.org/

data in different formats

Protein DB Sequence with ID

Protein ID

Homologous DB

Sequence with putative ID

SPIDER

No DB Sequence but no ID de novo

Multiple software

high confidence Sequence ID

inChorus

quantity Protein ID

PEAKS Q

PTMs Protein ID

PTM Finder

PEAKS Options 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.

De novo     Tools. 

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.

31 114 PRG +

+

+

+

30 115 PRG

29 116 PRG

28 117 PRG

Trypsin digest

[Reporter-Balance-Peptide] MS

-N H -N

H -N H -N

H

Mix MS MS/MS

117

116

115

114

Mass (m/z) 0 10 20 30 40 50 60 70 80 90 100

% In

tens

ity

72.0 509.8 947.6 1385.4 1823.2 2261.0 Mass (m/z)

0 10 20 30 40 50 60 70 80 90

100

% In

tens

ity

QGQPIGLGEASNDTWITTK

Charged Neutral loss

Isobaric Tag (Total mass = 145)

Reporter Balance Peptide Reactive Group

maxquant Quan5ta5ve  Tool 

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. 

MRM Targeted  Proteomics 

Quantitative Proteomics Results Prediction

Choose and Optimize Transistions

Selectivity, Sensitivity and Dynamic Range…

Tranche   Data Dissemina5on 

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…

Questions ? Pratik Jagtap

pratik@msi.umn.edu http://twitter.com/pratikomics

Sequest X! tandem OMSSA MaxQuant ProteinPilot Mascot Scaffold PEAKS TPP Tranche Trans-proteomic Pipeline Pipeline ProTIP

http://sitemaker.umich.edu/iwsmoi