protein sequence databases - cbs.umn.edu · fasta protein sequence •name and origin •&asta...

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Protein Sequence Databases

…and your Mass Spectrometry-based Proteomics Experiment



Outline

• Protein Database (DB) • Origin • Sources • Format • Size • Composition

• Selecting a database for mass spec search

• Effect of DB on mass spec search results

• Post MS analysis: protein annotation, ontology, alignment

Terminology

• FASTA

• Database repository

• NCBI database

• UniProtKB

• Swiss Prot

• Ref Seq (reference sequence)

• Homology

• Contaminants DB

• Ontology



FASTA Protein Sequence • Name and Origin

• FASTA (pronounced ‘fast-aye’)

• ORIGIN: for sequence similarity alignment tool (1985)

• REF: DJ Lipman, WR Pearson (1985) PMID: 2983426 "The algorithm has been implemented in a computer program designed to search protein databases very rapidly. For example, comparison of a 200-amino-acid sequence to the 500,000 residues in the National Biomedical Research Foundation library would take less than 2 minutes on a minicomputer, and less than 10 minutes on a microcomputer (IBM PC)."

• Stands for “fast all” – the file format worked with ‘all’ alphabets (amino acid and nucleotide)



FASTA Protein Sequence Format

• Structure: TEXT file

• Line 1: description line with sequence identifier

• Line 2: single amino acid letter protein sequence 80 characters wide

• Allowed characters: • AMINO ACID ONE-LETTER CODE • X • * • - • Custom one-letter amino acid codes



Line 1: description line with sequence identifier FASTA Format Header Line Sequence Identifiers



Line 2 FASTA Protein Sequence from NCBI- example

Line 1

Line 2

NOTE: In Sept 2016, gi numbers were replaced with accession.version identifiers



Selecting a Protein Sequence Database • Public repositories, such as

• NCBI • UniProtKB

• Swiss Prot: manually annotated and reviewed • TrEMBL: Automatically annotated and not reviewed

• Custom (from customer) • NOTE: format is important!

• Represent species (1 or more) from which protein sample originated • Example: Mouse protein expressed in E. coli

• Ideal size range ~ 2000 to < 1 million entries



Selecting a Protein Database: UniProtKB repository



Selecting a Protein Database: NCBI Ref Seq repository



Choose Your Taxonomy or Taxonomies

NOTES:

• If recombinant protein expressed in host cell, include host proteins & expressed protein(s)

• If protein database for your species has <2000 proteins, merge with another protein database (yeast) for statistical reasons

• Protein sequence headers must be parsed correctly



Taxonomy specification - UniProtKB

(19996)



Taxonomy specification - NCBI



Protein Database repository content for Thirteen-lined Ground Squirrel

Database Source Number of Proteins

Swiss-Prot* reviewed 20

TrEMBL* unreviewed 20,076

UniProt Reference Proteome 19,966

NCBI (‘non-redundant’) 30,130

NCBI Reference Sequence 29,842

* From UniProt



Protein Database Characteristics

…related to your mass spectrometry experiment



SPLICES FORM variants Sequence alignments: Protein Cytochrome P450 2D6



Protein Sequence Variants

SNP’s (single nucleotide polymporphisms)

https://hive.biochemistry.gwu.edu

Natural variants)



In silico trypsin digest, ‘native’ protein



In silico trypsin digest, with VARIANTS

1

2



Effect of Variant on Peptide Mass

Peptide example Peptide Mass * Peptide Sequence

1 – native 1730.8443 SELEEQLTPVAEETR

1 – variant (Q -> K) 1730.8806 SELEEKLTPVAEETR

1 – variant (Q -> K) 734.3566 SELEEK

1 – variant (Q -> K) 1015.5418 LTPVAEETR

2 – native 830.4366 EQVAEVR

2 – variant (V -> E) 860.4108 EQEAEVR

* Monoisotopic [M + H]+1



Proteomics Search Program Meets Protein Sequence Database • Protein sequence file is downloaded to local computer

• Merge with common lab contaminants (keratins and more) database • http://www.thegpm.org/crap/

• Protein database is imported or indexed in the proteomics search program (sequence format is critical)

• REVERSED sequences are generated for False Discovery Rate (FDR) calculations

• Protein sequences are digested with enzymes in silico



Database search > Protein List

• Database search algorithm matches spectrum > peptide > protein

• RESULTS: List of protein identifications with accession numbers

• POST Database search options (outside CMSP): 1. Protein annotation

2. Sequence alignment

3. Obtain related Gene Ontology information



POST Database search options

What you can do with your protein list.



1) Protein Annotation from UniProtKB



2) Sequence alignment with UniProt alignment tool



2) Sequence alignment with UniProt alignment tool: numerous amino acid labeling options

* (asterisk) indicates positions which have a single, fully conserved residue. : (colon) indicates conservation between groups of strongly similar properties - scoring > 0.5 in the Gonnet PAM 250 matrix. . (period) indicates conservation between groups of weakly similar properties - scoring =< 0.5 in the Gonnet PAM 250 matrix.



2) Sequence alignment with NCBI BLAST



3) Link Gene Ontology information to Proteins • Define: “The Gene Ontology (GO) project is a

collaborative effort to address the need for consistent descriptions of gene products across databases.”

• Ontologies/Vocabularies • molecular function: molecular activities of gene

products • cellular component: where gene products are active • biological process: pathways and larger processes made

up of the activities of multiple gene products

(http://geneontology.org/page/documentation)

http://geneontology.org/page/documentation



Molecular Function Pie Chart for a List of 96 Protein Identifiers (gi numbers) submitted to PANTHER (http://www.pantherdb.org/)

Protein list from Supplemental data REF: Thu TM et al (2016) Cell Reports, 15(6):1254-65; PMID: 27134171



Protein Class Pie Chart for a List of 96 Protein Identifiers (gi numbers) submitted to PANTHER (http://www.pantherdb.org/)




Biological Process Pie Chart for a List of 96 Protein Identifiers (gi numbers) submitted to PANTHER (http://www.pantherdb.org/)




Database Tools for Proteins

• http://geneontology.org/

• http://string-db.org/

• http://www.pantherdb.org/

• http://www.ingenuity.com/products/ipa (licensed at UM via MSI)

ALSO:

Match mass spec data to your RNA Seq data with:

• https://galaxyp.msi.umn.edu/

http://geneontology.org/

http://geneontology.org/

http://string-db.org/




http://www.pantherdb.org/

http://www.pantherdb.org/

http://www.ingenuity.com/products/ipa

http://www.ingenuity.com/products/ipa

protein sequence databases - cbs.umn.edu · fasta protein sequence •name and origin •&asta...

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