Protein Sequence Analysis - Protein Sequence Analysis - OverviewOverview

Raja MazumderRaja MazumderSenior Protein Scientist, PIRSenior Protein Scientist, PIR

Assistant Professor, Department of Biochemistry and Molecular Assistant Professor, Department of Biochemistry and Molecular BiologyBiology

Georgetown University Medical CenterGeorgetown University Medical Center

NIH Proteomics Workshop 2004

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OverviewOverview

ProteomicsProteomics and and protein bioinformatics protein bioinformatics (protein sequence analysis)(protein sequence analysis)

Why do protein sequence analysis? Why do protein sequence analysis? Searching sequence databasesSearching sequence databases Post-processing search resultsPost-processing search results Detecting remote homologsDetecting remote homologs

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Clinical Proteomics

From Petricoin et al., Nature Reviews Drug Discovery (2002) 1, 683-695From Petricoin et al., Nature Reviews Drug Discovery (2002) 1, 683-695

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Single protein and shotgun analysisSingle protein and shotgun analysis

Adapted from: McDonald et al. 2002. Disease Markers 18 99-105

Protein Bioinformatics

Mixture of proteinsG

el b

ased

sep

erat

ion

Single protein analysis

Digestion of protein mixture

Spot excisionand digestion

LC orLC/LC separation

Shotgun analysis

Peptides from a single protein

Peptides from many proteins

MS analysisMS/MS analysis

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Protein Bioinformatics: Protein Protein Bioinformatics: Protein sequence analysissequence analysis

Helps characterize protein sequences Helps characterize protein sequences inin silico silico and allows prediction of protein structure and and allows prediction of protein structure and functionfunction

Statistically significant BLAST hits Statistically significant BLAST hits usuallyusually signifies sequence homologysignifies sequence homology

Homologous sequences may or may not have Homologous sequences may or may not have the same function but would always (very few the same function but would always (very few exceptions) have the same structural foldexceptions) have the same structural fold

Protein sequence analysis allows protein Protein sequence analysis allows protein classification classification

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Development of protein sequence Development of protein sequence databasesdatabases

Atlas of protein sequence and structureAtlas of protein sequence and structure – – Dayhoff (1966) first sequence database (pre-Dayhoff (1966) first sequence database (pre-bioinformatics). Currently known as Protein bioinformatics). Currently known as Protein Information Resource (PIR)Information Resource (PIR)

Protein data bankProtein data bank (PDB) – structural database (PDB) – structural database (1972) remains most widely used database of (1972) remains most widely used database of structuresstructures

UniProtUniProt – The United Protein Databases – The United Protein Databases (UniProt, 2003) is a central database of protein (UniProt, 2003) is a central database of protein sequence and function created by joining the sequence and function created by joining the forces of the SWISS-PROT, TrEMBL and PIR forces of the SWISS-PROT, TrEMBL and PIR protein database activitiesprotein database activities

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Comparative protein sequence Comparative protein sequence analysis and evolutionanalysis and evolution

Patterns of conservation in sequences allows us Patterns of conservation in sequences allows us to determine which residues are under selective to determine which residues are under selective constraints (are important for protein function)constraints (are important for protein function)

Comparative analysis of proteins more sensitive Comparative analysis of proteins more sensitive than comparing DNAthan comparing DNA

Homologous proteins have a common ancestorHomologous proteins have a common ancestor Different proteins evolve at different ratesDifferent proteins evolve at different rates Protein classification systems based on Protein classification systems based on

evolution: evolution: PIRSFPIRSF and and COGCOG

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PIRSF and large-scale functional PIRSF and large-scale functional annotation of proteinsannotation of proteins

PIRSF structure is in the PIRSF structure is in the form of a network form of a network classification system classification system based on the evolutionary based on the evolutionary relationships of whole relationships of whole proteins and domainsproteins and domains

As part of the UniProt As part of the UniProt project, PIR has developed project, PIR has developed this classification strategy this classification strategy to assist in the propagation to assist in the propagation and standardization of and standardization of protein annotationprotein annotation

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Comparing proteinsComparing proteins

Amino acid sequence of protein generated Amino acid sequence of protein generated from proteomics experimentfrom proteomics experiment

e.g. protein fragment e.g. protein fragment DTIKDLLPNVCAFPMEKGPCQTYMTRWFFNFETGECELFAYGGCGGNSNNFLRKEKCEKFDTIKDLLPNVCAFPMEKGPCQTYMTRWFFNFETGECELFAYGGCGGNSNNFLRKEKCEKFCKFTCKFT

Amino-acids of two sequences can be Amino-acids of two sequences can be aligned and we can easily count the aligned and we can easily count the number of identical residues (or use an number of identical residues (or use an index of similarity) to find the % similarity.index of similarity) to find the % similarity.

Proteins structures can be compared by Proteins structures can be compared by superimpositionsuperimposition

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Protein sequence alignmentProtein sequence alignment

Pairwise alignmentPairwise alignment aa bb a a cc dd aa bb _ _ cc dd

Multiple sequence alignment usually provides Multiple sequence alignment usually provides more informationmore information a a bb a a cc d d a a bb _ _ cc d d x x bb a a cc e e

Multiple alignment difficult to do for distantly Multiple alignment difficult to do for distantly related proteins related proteins

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Protein sequence analysis Protein sequence analysis overviewoverview

Protein databasesProtein databases PIR and UniProtPIR and UniProt

Searching databasesSearching databases Peptide search, BLAST search, Text searchPeptide search, BLAST search, Text search

Information retrieval and analysisInformation retrieval and analysis Protein records at UniProt and PIRProtein records at UniProt and PIR Multiple sequence alignmentMultiple sequence alignment Secondary structure predictionSecondary structure prediction Homology modelingHomology modeling

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Universal Protein KnowledgebaseUniversal Protein Knowledgebase(UniProt) (UniProt)

PIR (Protein Information Resource)PIR (Protein Information Resource) has recently joined forces with EBI (European Bioinformatics Institute) and has recently joined forces with EBI (European Bioinformatics Institute) and SIB (Swiss Institute of Bioinformatics) to establish the UniProtSIB (Swiss Institute of Bioinformatics) to establish the UniProt

Literature-Based Annotation

UniProt Archive

UniProt NREF

Swiss-Prot

PIR-PSDTrEMBL RefSeq GenBank/EMBL/DDBJ

EnsEMBL PDB PatentData

Other Data

UniProt Knowledgebase

Classification

Automated Annotation

Clustering at 100, 90, 50%

Literature-Based Annotation

UniProt Archive

UniProt NREF

Swiss-Prot

PIR-PSDTrEMBL RefSeq GenBank/EMBL/DDBJ

EnsEMBL PDB PatentData

Other Data

UniProt Knowledgebase

Classification

Automated Annotation

Clustering at 100, 90, 50%

http://www.uniprot.org/

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Peptide SearchPeptide Search

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Query SequenceQuery Sequence

Unknown sequence is Q9I7I7Unknown sequence is Q9I7I7 BLAST Q9I7I7 against the UniProt BLAST Q9I7I7 against the UniProt

knowledgebaseknowledgebase ( (http://http://

www.pir.uniprot.org/search/blast.shtmlwww.pir.uniprot.org/search/blast.shtml)) Analyze resultsAnalyze results

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BLAST resultsBLAST results

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Text SearchText Search

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Text search results: display Text search results: display optionsoptions

Moving Pubmed ID and PDB ID into “Columns in Display”

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Text search results: add input Text search results: add input boxbox

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Text Search Result with NULL/NOT Text Search Result with NULL/NOT NULLNULL

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UniProt protein recordUniProt protein record: :

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SIR2_HUMAN protein recordSIR2_HUMAN protein record

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Are Q9I7I7 and SIR2_HUMAN Are Q9I7I7 and SIR2_HUMAN close homologs?close homologs?

Check BLAST resultsCheck BLAST results

Check pairwise alignmentCheck pairwise alignment

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Protein structure predictionProtein structure prediction

Programs can predict Programs can predict secondary structure secondary structure information with 70% information with 70% accuracyaccuracy

Homology modeling - Homology modeling - prediction of ‘target prediction of ‘target structure from closely structure from closely related ‘template’ related ‘template’ structurestructure

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Secondary structure predictionSecondary structure predictionhttp://bioinf.cs.ucl.ac.uk/psipred/http://bioinf.cs.ucl.ac.uk/psipred/

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Secondary structure prediction Secondary structure prediction resultsresults

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Sir2 Homolog-Nad ComplexSir2 Homolog-Nad Complex

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Homology modelingHomology modelinghttp://www.expasy.org/swissmod/SWISS-MODEL.htmlhttp://www.expasy.org/swissmod/SWISS-MODEL.html

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Homology model of Q9I7I7Homology model of Q9I7I7

Blue - excellentGreen - so soRed - not good

Yellow - beta sheetRed - alpha helixGrey - loop

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Sequence features: Sequence features: SIR2_HUMANSIR2_HUMAN

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Multiple sequence alignmentMultiple sequence alignment

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Multiple sequence alignmentMultiple sequence alignment

Q9I7I7, Q82QG9, SIR2_HUMANQ9I7I7, Q82QG9, SIR2_HUMAN

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Sequence features: Sequence features: CRAA_RABITCRAA_RABIT

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Identifying remote homologsIdentifying remote homologs

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Structure guided sequence Structure guided sequence alignmentalignment