August 20th 2015 Pratik Jagtapcbs.umn.edu/sites/cbs.umn.edu/files/public/downloads/08...Pratik Jagtap Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279

Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279

© 2015 Regents of the University of Minnesota. All rights reserved.

PROTEOINFORMATICS OVERVIEW

Center for Mass Spectrometry and Proteomics

August 20th 2015 Pratik Jagtap

http://www.cbs.umn.edu/msp



Outline

•  PROTEOMICS WORKFLOW •  PEAKLIST PROCESSING •  Search Databases Overview •  Protein Identification •  Protein Validation and Quantification •  Publication Guidelines

Terminology •  RAW file

•  Peaklist

•  Peaklist processing

•  Peptide-Spectral Match (PSM)

•  Genome Assembly and annotation

•  Variety of search databases



PROTEOMICS WORKFLOW

Eng et al 2011 Mol Cell Proteomics. 10(11): R111.009522.



Mass spectral data (.RAW)

StaAsAcal validaAon of Protein IdenAficaAon.

Protein IdenAficaAon

Processing Mass Spectrometer

PROTEOMICS WORKFLOW

Search databases Protein

QuanAtaAon.



Outline •  PROTEOMICS WORKFLOW

•  PEAKLIST PROCESSING •  Search Databases Overview •  Protein Identification •  Protein Validation and Quantification •  Publication Guidelines


•  Peaklist






© 2015 Regents of the University of Minnesota. All rights reserved. .

MASS SPECTRAL DATA



Cappadona et al 2012 Amino Acids. Sep 2012; 43(3): 1087–1108


MASS SPECTRAL DATA




PROTEOMICS WORKFLOW



Peaklist Processing



RAW DATA CONVERSION TOOLS

.RAW XRawfile library from

ThermoFinnigan Xcalibur software.

ReAdW

mzxML

http://tools.proteomecenter.org/wiki/index.php?title=Software:ReAdW

msconvert

ProteoWizard

mzML

http://proteowizard.sourceforge.net/

Others Raw2MSM extract_msn DeconMSn DTASuperCharge



Average ppm and Standard deviation improves when MaxQuant processed files are used.

ORBITRAP: PROCESSING AND EFFECTS



Peaklist Processing




PROTEOMICS WORKFLOW



Outline •  PROTEOMICS WORKFLOW •  PEAKLIST PROCESSING

•  Search Databases Overview

•  Protein Identification •  Protein Validation and Quantification •  Publication Guidelines


•  Peaklist







Mass spectral data (.RAW)

StaAsAcal validaAon of Protein IdenAficaAon.

Protein IdenAficaAon

Processing Mass Spectrometer

PROTEOMICS WORKFLOW

Search databases Protein

QuanAtaAon.



Search against database. Mass spectrum

DATABASE SEARCH



Salzberg Genome Biology 2007 8:102 doi:10.1186

DNA → GENOME → PROTEOMIC DATABASE.



GENOMIC AND PROTEOMIC DATABASES

Finished and Published Genomes •  3551 Bacterial Genomes. •  211 Archaeal Genomes. •  58 Eukaryal Genomes. •  3363 Viral Genomes

http://www.genomesonline.org/index



PROTEOMIC DATABASES

CUSTOMIZED DATABASES



Swiss-Prot is the manually annotated and reviewed section of the UniProt Knowledgebase (UniProtKB).

It is a high quality annotated and non-redundant protein sequence database,

which brings together experimental results, computed features and scientific

conclusions. http://en.wikipedia.org/wiki/Swiss-Prot

TrEMBL contains high-quality computationally analyzed records, which are enriched with automatic annotation.

The translations of annotated coding sequences in the EMBL-Bank/GenBank/DDBJ nucleotide sequence database are automatically processed and entered in

TrEMBL. http://en.wikipedia.org/wiki/TrEMBL

PROTEOMIC DATABASES



UNIPROT DATABASE



UNIPROT DATABASE



The Reference Sequence (RefSeq) collection provides a comprehensive, integrated, non-redundant, well-annotated set of sequences, including genomic DNA, transcripts, and proteins. RefSeq sequences form a foundation for medical, functional, and diversity studies. They provide a stable reference for genome annotation, gene identification and characterization, mutation and polymorphism analysis (especially RefSeqGene records), expression studies, and comparative analyses. http://www.ncbi.nlm.nih.gov/refseq/

PROTEOMIC DATABASES



CUSTOMIZED PROTEOMIC DATABASES

Customized database

repositories (CPTAC / UniMesh)

Genomic DNA

sequences.

Expressed sequence

tags / cDNA sequences.

Six-frame translation

Three-frame translation

Metagenomic databases.

Translation

RNASeq data.

Translation and database reduction

workflows

Proteomic databases.

24




PROTEOMICS WORKFLOW



Outline •  PROTEOMICS WORKFLOW •  PEAKLIST PROCESSING •  Search Databases Overview

•  Protein Identification

•  Protein Validation and Quantification •  Publication Guidelines


•  Peaklist





Documents

August 20th 2015 Pratik Jagtapcbs.umn.edu/sites/cbs.umn.edu/files/public/downloads/08...Pratik Jagtap Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279