Protein Sequence Databases …and your Mass Spectrometry-based Proteomics Experiment © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Outline Terminology • Protein Database (DB) • FASTA • Origin • Database repository • Sources • Format • NCBI database • Size • UniProtKB • Composition • Swiss Prot • Selecting a database for mass spec search • Ref Seq (reference • Effect of DB on mass spec sequence) search results • Homology • Post MS analysis: protein • Contaminants DB annotation, ontology, alignment • Ontology © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 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) © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 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 © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Line 1: description line with sequence identifier FASTA Format Header Line Sequence Identifiers © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Line 2 FASTA Protein Sequence from NCBI- example Line 1 Line 2 NOTE: In Sept 2016, gi numbers were replaced with accession.version identifiers © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 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 © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Selecting a Protein Database: UniProtKB repository © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Selecting a Protein Database: NCBI Ref Seq repository © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 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 © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Taxonomy specification - UniProtKB (19996) © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Taxonomy specification - NCBI © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 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 © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Protein Database Characteristics …related to your mass spectrometry experiment © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 SPLICES FORM variants Sequence alignments: Protein Cytochrome P450 2D6 © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Protein Sequence Variants Natural variants) SNP’s (single nucleotide polymporphisms) https://hive.biochemistry.gwu.edu © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 In silico trypsin digest, ‘native’ protein © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 In silico trypsin digest, with VARIANTS 1 2 © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 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 © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 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 © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 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 © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 POST Database search options What you can do with your protein list. © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 1) Protein Annotation from UniProtKB © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 2) Sequence alignment with UniProt alignment tool © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 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. © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 2) Sequence alignment with NCBI BLAST © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 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) © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 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,