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IntroductionLarge-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 an 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.

The iProXpress Knowledge System for Proteomic Data AnalysisZhang-Zhi Hu1; Hongzhan Huang1; Peter McGarvey1; An Chi2; Julio Valencia3; Cathy H. Wu1

1Department of Biochemistry and Molecular & Cellular Biology, Georgetown University Medical Center, Washington, DC 20007; 2 University of Virginia Department of Chemistry, Charlottesville, VA 22904; 3Laboratory of Cell Biology, National Cancer Institute, Bethesda, MD 20892

Protein Information

Matrix

iProXpress

integrated Protein eXpression Analysis System

Functional Annotation

Expression Profiling

Protein Mapping

IP/2D/MS Proteomic DataGene Expression

Gene/Protein ID list Peptide Sequence

UniProt

iProClass

Function Categorization Chart

Two-Way Comparison Matrix

Pathway MapInteraction Map

GO tree visualization

Protein Information Matrix

Figure 1. An Overview of the iProXpress System

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.

Conclusion

The prototype iProXpress system enables the analysis of large-scale expression data through its functionalities in ID/peptide mapping, sequence analysis, functional annotation, and functional profiling. The case study on melanosome proteome analysis illustrates that the use of the system greatly facilitates the study of melanosome biogenesis and pigmentation related human diseases. The system should be applicable to a broad range of genomic and proteomic profiling studies.

Sorting of data sets and display of protein information matrix

Functional categorization based on gene ontology

Two-way comparative matrix

NIAID Biodefense Proteomics Program Mater catalog of proteins from NIAID Biodefence Proteomic Research Centers.

A catalog of proteins from Harvard University, cDNA clones of which are used for protein chip assays to detect protein-protein interactions in situ.

Browsing selected complete proteome(s) with protein links to the Proteomic Center data.

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 2Q8IWB8 : CCR4-NOT transcription complex, subunit 1, isoform bP27449 : Vacuolar ATP synthase 16 kDa proteolipid subunit (EC 3.6.3.14) (OMIM: 108745)Q71RS6 : Ion transporter JSX [Homo sapiens] – human skin color geneQ6ZTT7 : Hypothetical protein FLJ44232 [Homo sapiens]Q16444 : Phosphoglycerate kinase (Fragment) [Homo sapiens] (47 aa)

Table 1. Mapping of mouse color genes to human melanosome proteins Table 2. Partial list of stage-specific melanosome proteins

Figure 2. Electron microscopy images showing the morphological characteristics of the melanosomes during maturation. In pigmented cells such as MNT1, stage I melanosomes are relatively spherical organelles with an amorphous matrix; at Stage II, organelles elongate, the internal fibrils form regular parallel arrays and melanin synthesis starts at granular sites studding the internal walls of the melanosome; at late-stage stage III, deposition of melanin on the melanosomal matrix becomes evident; till stage IV, the organelle is fully matured with a heavy accumulation of melanin. For those of non-pigmented mutant cells such as SkMel-28, melanosomes stay at early developmental stages and melanin synthesis is halted due to the dysfunctional maturation and a large number of early stage melanosomes are accumulated as demonstrated on the panels to the right.

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)

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 Sec8P46459 : Vesicle-fusing ATPase (EC 3.6.4.6) (interact)P14415 : Sodium/potassium-transporting ATPase beta-2 chain (TM)

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-13A2Transport: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)

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 (=mouse Rab38)P51810 : G-protein coupled receptor 143 (=mouse Gpr143) P53794 : Sodium/myo-inositol cotransporter (Na(+)/myo-inositol cotransporter)

Slominski, A. et al. Physiol. Rev. 84: 1155-1228 2004; Copyright ©2004 American Physiological Society

Figure 3B. Schematic of melanosomal proteins (Hypothetical depiction)

Gene Symbol

Murine LocusFunction in

PigmentationHuman Melanosome

ProteinsHuman Disease (OMIM)

Ednrb piebald spotting (s)melanoblast differentiation

P24530 : ET-B (Common stage1& MNT1 stage2)

Waardenburg-shah syndrome [277580]…

Sfxn1 flexed tailTricarboxylate carrier

Q9H9B4 : Sideroflexin-1(Common early stage)

*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]…

*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]

*Gpnmbiris pigment dispersion (ipd)

Apparent melanosomal component

Q14956 : Transmembrane glycoprotein NMB precursor (Common all stage)

Glaucoma-related pigment dispersion syndrome-1 [604368 ]

*Matp underwhite (uw) transporter

Q9UMX9 : Melanoma antigen AIM1 Q6P2P0 : Membrane-associated transporter protein, isoform b (MNT1 stage1 & 2)

OCA4 [606574]

Rab27a ashen (ash)melanosome transport

Q6IAS8 : RAB27A proteinP51159 : Ras-related protein Rab-27A (Common all stage)

Griscelli syndrome, type 2 [607624]

gdn golden (gdn)

Causes delayed and reduced development of melanin pigmentation

Q71RS6Ion transporter JSX (Unique late stage)

SLC24A5, a human skin color geneScience 16 December 2005: 1754-1755.

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/

Organelle # Protein reported

# Entries mapped

Common with melanosome *

References

Mouse ER (ER) ~141 131 57 (M:51, S:36)-19(33%) all stage

Knoblach et al, 2003

Human neuromelanin granules (NG)

72 72 43 (M:38, S:36)-22(51%) all stages

Tribl et al, 2005

Rat synaptosome (SY)

200 88 43 (M:35, S:27)-14(33%) all stages

Witzmann et al, 2005

Rat lysosome (LY) 215 116 49 (M:40, S:38)-13(27%) all stages

Bagshaw et al, 2005

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, 2004Proteins Present in at Least 3 Other Organelles in Either Early Stages or All Stages of

Melanosomes (1-ER, 2-NG, 3-SY, 4-EX, 5-PL, 6-LY)

1 2 3 4 5 6P30101 |+|+|+|+|+||+|+|-|+|-|-| Protein disulfide-isomerase A3 precursor P35232 |+|+|-|-|+||+|-|+|+|-|+| Prohibitin P60709 |+|+|+|-|-||+|-|+|+|-|-| Actin, cytoplasmic 1 (Beta-actin)Q5FVE7 |+|+|-|-|-||+|-|+|-|-|+| Voltage-dependent anion channel 1P04406 |+|+|+|+|+||-|+|+|-|-|+| Glyceraldehyde-3-phosphate dehydrogenaseP04843 |+|+|+|+|+||-|+|-|-|+|+| protein glycosyltransferaseP06576 |+|+|+|-|+||+|+|+|-|-|-| ATP synthase beta chain, mitochondrialP10809 |+|+|+|+|+||-|+|+|+|-|-| 60 kDa heat shock protein, mitochondrialP11142 |+|+|+|+|+||-|+|+|+|-|+| Heat shock cognate 71 kDa protein

* M – MNT1 cells ; S – Skmel28 cells; Percent of protein common in all stages of MNT1 melanososmes are also indicated. The complete listing of the common proteins is at http://pir.georgetown.edu/~huz/datamining/proteomics/ .

Table 3. Summary of the comparison of organellar proteomes

ER

trans-Golgi Network

cis-Golgi

Lysosome

AP3

Stage I

Stage IV

Stage III

Stage II

AP1

cell membrane

AP2

Early Endosome

Late Endosome

AP3

AP3APxAP3

AP4

Tyrgp100 (10%)

Gp100gp100

Tyr

AP3gp100

Tyrosinase

gp100 (90%)

Tyrp1, Dct and MART1 ?

Figure 3A. Trafficking and sorting of proteins to melanosomes. Hearing 2005.

Proposal: a list of proteins characteristic of melanasome that may consist of:

• previously known melanosome-specific proteins;

• current short list of melanosome stage-specific proteins as proposed in table 2;

• some constituent proteins commonly found in several other organelles such as those in table 3.

A Case Study – Organellar proteomes of various stages of melanosomes from human melanoma cell lines• Mapping to known mouse coat color genes led to identification of 17 essential human melanosome related proteins; • Identification of possible stage-specific melanosome proteins for validation; • Comparison of melanosome proteome with those of several other organelles.