Spring 2009 Intro Bioinformatiatics Proteomics Spring 2009 Bioinformatiatics

Spring 2009 Intro Bioinformatiatics Proteomics

Spring 2009 Bioinformatiatics workflow Proteomics Workflow • • • Sample Prep Sequencing Database Search Protein ID Protein Interactions

General workflow of proteomics analysis Proteins/peptides Digestion and/or separation External data sources taxonomy, ontologies, bibliography… Applications Systems biology (pathways, interactions. . ) biomarkerdiscovery, drug targets 2 D gel image aquisition and storage MALDI, MS/MS Identification PMF Quantification MS/MS DIGE LC-MS & Tags Store peak lists and all meta data

General workflow of proteomics analysis Proteins/pe ptides KEGG PDB DIP OMIM Reactome PROSIT Pfam SPIN BOND STRING Ami. GO David Pub. Med MEDLINE Digestion and/or separation Sequence data bases: Make 2 D 2 D Page data bases Swiss 2 D PAGE, Gelbank, Cornelia, Word. PAGE EMBL Nucleotide Sequence Database Gen. Bank Uni. Prot. KB/Swiss-Prot & Tr. EMBL Ensemble Mascot EST database PIR MALDI, MS/MS Identification Quantification Imaging tools: Melanie, PDQuest Progenesis Delta 2 D Sequest Storing/ organising: Aldente Proteincsape Popitam MSight Phenyx Find. Mod Profound Pep. Frag MS-Fit OMSSA Search XLinks

General workflow of proteomics analysis Proteins/pe ptides Digestion and/or separation 2 D gel databases: Data integration on the web Image data and textual information 2 D Page data Imaging Softwares: bases Make 2 D The ability to compare two gels (images) and then identify differently expressed spots • Melanie • Swiss 2 D PAGE • PDQuest • Gelbank • Progenesis • Cornelia • Delta 2 D • Word. PAGE Proteinscape –platform for storing, organizing data MSight -representation of mass spectra along with data from the separation

Spring 2009 Bioinformatiatics Laser-Capture Micro dissection, LMC Laser capture Technique for selectively sampling certain cells within a tissue Biopsy Transfer film Tissue sample Tumor Glass slide Laser beam activates film Cells Selected cells are transferred Genomic/proteomic analysis Modified from “National Cancer Institute”, US National Institutes of Health: http: //www. cancer. gov/cancertopics/understandingcancer/moleculardiagnostics/Slide 29

Spring 2009 Bioinformatiatics Template

Spring 2009 Bioinformatiatics Fractionation Affinity Purification

Spring 2009 Bioinformatiatics 2 D gels at Swissprot 2 D Electrophoresis Swissprot Ex. Pa. Sy Database

Spring 2009 Bioinformatiatics Template Protein Digestion • Primary sequence must be accessible • Denature – urea in solution or SDS in gel • Reduce & alkylate disulfide bonds between cysteines • dithiothreitol (DTT) & Iodoacetamide (IAA) • Digest with enymes • Purify peptide fragments

Spring 2009 Bioinformatiatics Template

Spring 2009 Bioinformatiatics codon Usage Standard Genetic Code (transl_table=1) AAs = FFLLSSSSYY**CC*WLLLLPPPPHHQQRRRRIIIMTTTTNNKKSSRRVVVVAAAADDEEGGGG Starts = ---M---------------M--------------Base 1 = TTTTTTTTCCCCCCCCAAAAAAAAGGGGGG Base 2 = TTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGGTTTTCCCCAAA Base 3 = TCAGTCAGTCAGTCAGTCAGTCAGTCAGTCA The. Bacterial Ciliate. Hexamita and Plant. Nuclear Plastid Code (transl_table=6) (transl_table=11) AAs == FFLLSSSSYYQQCC*WLLLLPPPPHHQQRRRRIIIMTTTTNNKKSSRRVVVVAAAADDEEGGG AAs FFLLSSSSYY**CC*WLLLLPPPPHHQQRRRRIIIMTTTTNNKKSSRRVVVVAAAADDEEGGGG Starts = ------------------M--------------Starts Base 1 ==---M------------MMMM--------M------TTTTTTTTCCCCCCCCAAAAAAAAGGGGGG Base 1 == TTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGGTTTTCCCCAAA Base 2 TTTTTTTTCCCCCCCCAAAAAAAAGGGGGGGG Base 3 = TCAGTCAGTCAGTCAGTCAGTCAGTCAGTCA G Base 2 =

Spring 2009 Bioinformatiatics Unusual amino acids Unusual Amino Acids

Spring 2009 Bioinformatiatics phosphorylation Phosphorylation - signal transduction m. RNA

Spring 2009 Bioinformatiatics Template

MS analysis

Antibody arrays Good for low-abundance proteins Problem is antibody specificity

Array-based protein interaction detection

Protein microarrays

Yeast Two-Hybrid System

How to organize information? • Gene Ontology – Biological process • Frequently from biochemical analyses • In silico analysis – Molecular function • Biochemical analysis – Cellular component • Biochemical analysis • GFP or other tagging

Interaction maps - Grid

challenges • Complexity – some proteins have >1000 variants • Need for a general technology for targeted manipulation of gene expression • Limited throughput of todays proteomic platforms • Lack of general technique for absolute quantitation of proteins

Protein Profiling Measure the expression of a set of proteins in two samples and compare them - Comparative proteomics • 2 D gel electrophoresis • Difference gel electrophoresis (DIGE) • LC-MS/MS using coded affinity tagging (ICAT, i. Trac, SILAC. . ) • Protein. Chip Array (SELDI analysis) • Antibody arrays