august 11th 2016 pratik jagtap - college of biological … jagtap ©2016 regents of the university...

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 11th 2016

Pratik Jagtap

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

©2016 Regents of the University of Minnesota, All rights reserved.





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)

Statistical validation of Protein Identification.

Protein Identification

Processing Mass Spectrometer

PROTEOMICS WORKFLOW

Search databases Protein

Quantitation.




Outline

• PROTEOMICS WORKFLOW

• PEAKLIST PROCESSING • Search Databases Overview

• Protein Identification



Terminology

• RAW file

• 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


http://omics.pnl.gov/software/DeconMSn.php

http://omics.pnl.gov/software/DeconMSn.php



Average ppm and Standard deviation

improves when MaxQuant processed

files are used.

ORBITRAP: PROCESSING AND EFFECTS




Peaklist Processing





PROTEOMICS WORKFLOW




Outline


• PEAKLIST PROCESSING

• Search Databases

Overview • Protein Identification



Terminology

• RAW file

• Peaklist








Mass spectral data (.RAW)

Statistical validation of Protein Identification.

Protein Identification

Processing Mass Spectrometer

PROTEOMICS WORKFLOW

Search databases Protein

Quantitation.




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




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


• PEAKLIST PROCESSING

• Search Databases Overview

• Protein

Identification • Protein Validation and Quantification


Terminology

• RAW file

• Peaklist






august 11th 2016 pratik jagtap - college of biological … jagtap ©2016 regents of the university...

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