The iProXpress Knowledge System for Proteomic Data Analysis

1. Introduction Large-scale proteomic profiling of biological samples such as cells, organelles or biological fluids has led to discovery of numerous key and novel proteins involved in many biological/disease processes, as well as to the identification of novel disease biomarkers and potential therapeutic targets. Bioinformatics infrastructure and systems play instrumental roles in such data analyses and discovery processes. iProXpress (integrated Protein eXpression system) is a prototype analysis system designed to help analyze proteomic and genomic data, such as protein/peptide and gene profiles from MS proteomic and microarray gene expression experiments. It has been applied to several studies including the expression profile analysis of hormone-induced changes in tumor cells, and is currently being adopted for analyses of pathogen/host genomic and proteomic data produced from the NIAID Biodefense Proteomic Program. We will present a case study where proteomes of various stages of melanosomes from human melanoma cells were analyzed using the iProXpress system to illustrate its utility in facilitating a better understanding of pathways of melanin synthesis and melanosome biogenesis. • System Designs • iProXpress consists of three major components: • The PIR (Protein Information Resource) data warehouse with integrated protein information, • Analytical tools for sequence analysis and functional annotation, and • Graphical user interface for categorization and visualization of expression data. • Major Functionalities • Gene/Peptide to Protein Mapping. Gene or protein lists are mapped to corresponding entries in UniProt Knowledgebase (UniProtKB) of all known proteins based on gene/protein IDs, names or sequences. • Protein information matrix. A comprehensive matrix is generated, summarizing salient features including gene ontology (GO) and pathways retrieved from underlying PIR protein databases with annotated experimental literature information or inferred based on sequence similarity. • Protein Data Analysis. By iterative categorization and sorting of proteins in the information matrix, users can correlate expression/interaction patterns to protein properties for pathway and network discovery. • Melanosome Proteome Analysis • ID mapping. Total 2298 gi numbers were mapped using UniProt/PIR ID mapping service, which converts common gene/protein IDs (e.g., gi number) to UniProtKB AC/ID and vice versa. 1253 were mapped to UniProtKB sequences. • Peptide mapping. Peptides from MS data were matched against all human sequences in UniProtKB with a two-step procedure: direct mapping or mapping using UniRef90 clusters (90% or more sequence identity grouped in the cluster), giving 1506 mapped proteins. Combining both ID and peptide mappings, 1936 (84%) of the proteins were mapped to 1532 UniProtKB sequences. • Protein information matrix. A comprehensive protein information matrix were generated from underlying PIR data warehouse (UniProt/iProClass) or inferred based on sequence homologies for melanosome proteins. Attributes in the matrix include protein name, family, domain/motif/site, isoform, post-translational modification, GO, function/functional category, structure/structural classification, pathway, protein interaction and complex, etc. • Melanosome proteome analysis: The melanosome proteome datasets were partitioned into 12 groups and categorization and sorting functions for each group or all datasets were provided based on the protein information matrices especially of gene ontologies (GO) and pathways (KEGG and BioCarta). Iterative categorization and sorting of proteins based on functions, pathways, and/or other attributes were carried out to generate various protein clusters, from which interesting unique or common proteins at different stages of melanosome biogenesis can be identified in combination with manual examination. • Comparative analysis of organelle proteomes. Melanosome proteomes of early or late stages were compared with other organelle proteomes such as lysosome, synaptosome and endosome. This comparative analysis coupled with other bioinformatic analysis was aimed at deducing a set of signature proteins characteristics of the melanosme. The iProXpress Knowledge System for Proteomic Data Analysis Hongzhan Huang,Zhang-Zhi Hu, Peter McGarvey and Cathy H. Wu Protein Information Resource, Georgetown University, Washington, DC 20007 Figure 1. An Overview of the iProXpress System Gene Expression IP/2D/MS Proteomic Data iProXpress integrated Protein eXpression Analysis System Gene/Protein ID list Peptide Sequence UniProt Protein Mapping iProClass Functional Annotation Protein Information Matrix Expression Profiling Protein Information Matrix GO tree visualization Interaction Map Pathway Map Function Categorization Chart Two-Way Comparison Matrix Sorting of data sets and display of protein information matrix NIAID Biodefense Proteomics Program Functional categorization based on Gene Ontology Master Protein Diretory from NIAID Biodefence Proteomic Research Centers. Two-way comparative matrix Browsing selected complete proteome(s) with protein links to the Proteomic Center data. Table 1. Mapping of mouse color genes to human melanosome proteins Table 2. Partial list of stage-specific melanosome proteins Gene Symbol Murine Locus Function in Pigmentation Human Melanosome Proteins Human Disease (OMIM) MNT1 Stage I (77) P36955 : Pigment epithelium-derived factor precursor (PEDF) Q14254 : Flotillin-2 (Epidermal surface antigen) P07093 : Glia derived nexin precursor (GDN) P24390 : ER lumen protein retaining receptor 1 (KDEL receptor 1) (TM) O14880 : Microsomal glutathione S-transferase 3 (TM) Ednrb piebald spotting (s) melanoblast differentiation P24530 :ET-B (Common stage1 & MNT1 stage2) Waardenburg-shah syndrome [277580]… Sfxn1 flexed tail Tricarboxylate carrier Q9H9B4 : Sideroflexin-1(Common early stage) MNT1 Stage II (112) Q12846 : Syntaxin-4 (TM) (interact with O75379 Vesicle-associated membrane protein 4; Q15836 Vesicle-associated membrane protein 3) Q04656 : Copper-transporting ATPase 1 (=mouse Atp7a) Adaptor proteins: O95782: AP-2 (~mouse Ap3bl) (also Skmel) Q96EL6: Adaptin (~mouse Ap3d) Vacuolar protein sorting: Q96A65 : Exocyst complex component Sec8 P46459 : Vesicle-fusing ATPase (EC 3.6.4.6) (interact) P14415 : Sodium/potassium-transporting ATPase beta-2 chain (TM) *Rab38 chocolate (cht) Targeting of Tyrp1 protein to the melanosome P57729 : Ras-related protein Rab-38. (uniqueSkmel28) 606281 *si silver (si) melanosomal matrix protein P40967 : Pmel 17 precursor (Common all stage) Oculocutaneous albinism [155550] *Tyr albino, color (c) melanogenic enzyme P14679 : Tyrosinase precursor (Unique MNT1) OCA1 [203100]; OCA1B [606952]… MNT4 Stage IV (267) Adaptor protein: Q9Y6Q5 : Adaptor protein complex AP-1 mu-2 subunit, (interact with P63010: AP2B1) Motor poteins: Q14203 : Dynactin-1 (Progressive lower motor neuron disease [OMIM:607641]) (interact with P18669: Phosphoglycerate mutase 1) Q9H193 : KINESIN-13A2 Transport: Q99747 : Gamma-soluble NSF attachment protein (also Skmel) Q99698 : Lysosomal trafficking regulator (=mouse Lyst) Vacuolar protein sorting: Q9H444 : VPS32 (~mouse Vps33a) (interact) (also Skmel) Q9NZZ3 : VPS60 (~mouse Vps33a) *Tyrp1 brown (b) melanosomal enzyme/stabilizing factor P17643 : 5,6-dihydroxyindole-2-carboxylic acid oxidase precursor. (Unique MNT1) Rufous albinism, ROCA [115501]; OCA3 [203290]; Precocious graying of hair [278400] A Case Study Table 3. Summary of the comparison of organellar proteomes *Gpnmb iris pigment dispersion (ipd) Apparent melanosomal component Q14956 : Transmembrane glycoprotein NMB precursor (Common all stage) Glaucoma-related pigment dispersion syndrome-1 [604368 ] Organelle # Protein reported # Entries mapped Common with melanosome * References • Organellar proteomes of various stages of melanosomes from human melanoma cell lines • Mapping to known mouse coat color genes led to identification of 17 human melanosome related proteins; • Identification of possible stage-specific melanosome proteins for validation; • Comparison of melanosome proteome with those of several other organelles. Mouse ER (ER) ~141 131 57 (M:51, S:36) -19(33%) all stage Knoblach et al, 2003 *Matp underwhite (uw) transporter Q9UMX9 : Melanoma antigen AIM1 Q6P2P0 : Membrane-associated transporter protein, isoform b (MNT1 stage1 & 2) OCA4 [606574] Skmel28 unique (143) Both melanosome-specific proteins Tyrosinase and TYRP1 are absent in Skmel28 data set, suggesting that their absence can partially account for the lack of melanin production in Skmel28. P57729 : Ras-related protein Rab-38 (=mouseRab38) P51810 : G-protein coupled receptor 143 (=mouseGpr143) P53794 : Sodium/myo-inositol cotransporter (Na(+)/myo-inositol cotransporter) Human neuromelanin granules (NG) 72 72 43 (M:38, S:36) -22(51%) all stages Tribl et al, 2005 Rab27a ashen (ash) melanosome transport Q6IAS8 : RAB27A protein P51159 : Ras-related protein Rab-27A (Common all stage) Griscelli syndrome, type 2 [607624] Rat synaptosome (SY) 200 88 43 (M:35, S:27) -14(33%) all stages Witzmann et al, 2005 All stage IV-specific membrane protein: P50443 : Sulfate transporter (Solute carrier family 26 member 2) (OMIM: 600972) Q9NZ45 : Protein C10orf70 P33121 : Long-chain-fatty-acid--CoA ligase 1 (LACS 1) (OMIM: 152425) (also in ER) Q8NCC2 : Hypothetical protein FLJ90355 (Solute carrier family 2 Q8IWB8 : CCR4-NOT transcription complex, subunit 1, isoform b P27449 : Vacuolar ATP synthase 16 kDa proteolipid subunit (EC 3.6.3.14) (OMIM: 108745) Q71RS6 : Ion transporter JSX [Homo sapiens] – human skin color gene Q6ZTT7 : Hypothetical protein FLJ44232 [Homo sapiens] Q16444 : Phosphoglycerate kinase (Fragment) [Homo sapiens] (47 aa) Rat lysosome (LY) 215 116 49 (M:40, S:38) -13(27%) all stages Bagshaw et al, 2005 gdn golden (gdn) Causes delayed and reduced development of melanin pigmentation Q71RS6Ion transporter JSX (Unique late stage) SLC24A5, a human skin color gene Science 16 December 2005: 1754-1755. Human platelet (PL) ~93 71 33 (M:26, S:22) -6(18%) all stages Martens et al, 2005 Human exosome (EX) ~56 55 42 (M:42, S:32) -18(43%) all stages Mears et al, 2004 This is a partial list of total 17 mapped genes. The others include Lyst, Ostm1, Dct, Atp7a, Gpr143, Myo5a and Krt2-17. For complete list, go to http://pir.georgetown.edu/~huz/datamining/proteomics/ Contact pirmail@georgetown.edu http://pir.georgetown.edu/iproxpress