pt iproteomics - university of north carolina at chapel hill · 2010-11-17 · the web for peptides...

P t iP t iProteomicsProteomics

Areas of Application for Proteomics in Toxicology

• Diagnostics:► detection of antigens and antibodies in blood samples► detection of antigens and antibodies in blood samples► profiling of sera to discover new disease markers► environment and food monitoring

• Protein expression profiling:► organ- and disease-specific arrays► mode of action analysis► mode of action analysis

• Protein functional analysis:► ligand-binding properties of receptors► enzyme activities► protein-protein interactions

Most Commonly Used Proteomics Techniques: Antibody arrays

Protein activity arrays

2-D gels

“Shotgun” proteomics

ICAT technology

SELDI

100% protein sequence coverage: a modern form of surrealism in proteomics. Meyer et al Amino Acids. 2010 Jul 13.

S i t i t i i t ti

Antibody Arrays

• Screening protein-protein interactions • Studying protein posttranslational modifications• Examining protein expression patternsg p p p

Antibody Arrays

The layout design of the BD Clontech™ Ab Microarray 380. The BDClontech™ Ab Microarray 380 (#K1847-1) contains 378 monoclonal antibodiesarrayed in a 32 x 24 grid. Each antibody is printed in duplicate. Dark gray dotsat the corners represent Cy3/Cy5-labeled bovine serum albumin (BSA) spots,which serve as orientation markers. The open circles correspond to unlabeledBSA spots, which serve as negative controls. For complete descriptions of theBSA spots, which serve as negative controls. For complete descriptions of theproteins profiled by the Ab Microarray 380, visit bdbiosciences.com

Limitations, Challenges and Bottlenecks

• Protein production: p►cell-based expression systems for recombinant proteins► purification from natural sources► production in vitro by cell-free translation systems► production in vitro by cell free translation systems► synthetic methods for peptides

• Immobilization surfaces and array formats:► Common physical supports include glass slides, silicon, ► Co o p ys ca suppo s c ude g ass s des, s co ,microwells, nitrocellulose or PVDF membranes, microbeads

• Protein immobilization should be:► reproducible► ep oduc b e► applicable to proteins of different properties (size, charge, …)► amenable to high throughput and automation, and compatible with retention of fully functional protein activity► such that maintains correct protein orientation

• Array fabrication:► robotic contact printing► ink-jetting► piezoelectric spotting► photolithography

Protein Activity Arrays

Panomics® Transcription Factor Arrays:

A set of biotin-labeled DNA binding oligonucleotides (TranSignal™ probe mix) is preincubated with any nuclear extract of interest to allow the formation of protein/DNA (or TF/DNA) complexes;

The protein/DNA complexes are separated from the free probes;

The probes in the complexes are then extracted and hybridized to theextracted and hybridized to the TranSignal™ Array. Signals can be detected using either x-ray film or chemi-luminescent imaging. All reagents for HRP-based h il i t d t ti i l d dchemiluminescent detection are included.

Source: Panomics, Inc.

Protein Activity Arrays

G l Shift A P t i AGel Shift Assay Protein Array

Source: Panomics, Inc.

2D Gel Electrophoresis + Mass Spectrometry

Meyer et al Amino Acids. 2010 Jul 13.

2D Gel Electrophoresis Protein Resolution

Bandara & Kennedy (2002)

2D Gel Electrophoresis Protein Resolution

Courtesy of Bio-Rad

Courtesy of Bio-RadCourtesy of Fermentas

2D Gel Electrophoresis Image Analysis

Courtesy of Decodon

Courtesy of Alphainnotech

2D Gel ElectrophoresisMass Spectrometry

Source: UNC Proteomics Core Facility

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SEQUEST is a program that uses raw peptide MS/MS data (off TSQ-7000 or LCQ) to identify

Imag

SEQUEST is a program that uses raw peptide MS/MS data (off TSQ 7000 or LCQ) to identifyunknown proteins. It works by searching protein and nucleotide databases (in FASTA format) onthe web for peptides that match the molecular weight of the unknown peptides produced bydigestion of your protein(s) of interest. Theoretical MS/MS spectra are then generated and ascore is given to each one. The top 500 scored theoretical peptides are retained and a crosscorrelation analysis is then performed between the un-interpreted MS/MS spectra (real MS/MScorrelation analysis is then performed between the un interpreted MS/MS spectra (real MS/MSspectra) of unknown peptides with each of the retained theoretical MS/MS spectra. Highlycorrelated spectra result in identification of the peptide sequences and multiple peptideidentification and thus determine the protein and organism of origin corresponding to theunknown protein sample.

SHOTGUN PROTEOMICS

Proteins are analyzed by standard shotgun proteomics, beginning with tryptic digest of a protein mixture, liquid chromatographic separation of the mixture (2D HPLC), analysis of peptide masses by mass spectrometry (MS) and fragmentation of peptides and subsequent analysis of the fragmentation spectra (MS/MS). Each step introduces bias into the peptides ultimately interpreted from the analysis, thereby affecting the probability pij of observing each peptide j from protein i. APEX involves training a classifier to estimate Oi, the prior estimate of the number of unique peptides expected from a given protein during such an experiment. By correcting for Oi, the number of peptides observed per protein thereby provides an estimate of the protein's abundance. HPLC, high‐performance liquid chromatography. Nature Biotechnology 25, 117 ‐ 124 (2007)

Isotope Coded Affinity Tag (ICAT) Analysis


SELDI Analysis

Perticoin et al., Toxicologic Pathology, 32(Suppl. 1):122–130, 2004

Representative “raw” spectra and“gel-view” (grey-scale) of serumgel view (grey scale) of serumfrom a normal donor, and frompatients with either BPH (benignprostate hyperplasia) or prostatecancer (PCA) using the IMAC3-Cu

From: www.evms.edu/vpc/seldi/seldiprocess/

cancer (PCA) using the IMAC3 Cuchip chemistry

The upregulated 11.9 kDa biomarker from the TMPD-treated rats was searched via Tagldent (SWISS-PROT), yielding a tentative identity as parvalbumin-alpha. This candidate was subsequently purified, peptide mapped and searched to confirm the identity. Parvalbumin is involved in muscle homeostasis.

Courtesy CIPHERGEN®

Limitations, Challenges and Bottlenecks

• Resolution: • Resolution: ► number of proteins that can be separated/distinguished (500,000?!?)► pI resolution► mass resolution (gels and mass spectrometry)

• Amount of the protein in the sample:• Amount of the protein in the sample:► too little to be seen on a 2D gel?► too little to be extracted and digested?

• Protein solubility• Database searching and peptide identification• Database searching and peptide identification


Schneider LV, Hall MP. Drug Discov Today. 2005 10:353-63.

T di i l l t h ti l i f t li t t l t i Th t i t d H 3 10Two-dimensional electrophoretic analysis of rat liver total proteins. The proteins were separated on a pH 3–10nonlinear IPG strip (left), or pH 4-7 IPG strip (right), followed by a 10% SDS–polyacrylamide gel. The gel wasstained with Coomassie blue. The spots were analyzed by MALDI-MS. The proteins identified are designatedwith the accession numbers of the corresponding database.

From Fountoulakis & Suter (2002)

Two dimensional electrophoretic analysis of rat liver cytosolic proteins The proteins were separated on a pHTwo-dimensional electrophoretic analysis of rat liver cytosolic proteins. The proteins were separated on a pH 3–10 nonlinear IPG strip (left), or pH 5–6 IPG strip (right), followed by a 10% SDS–polyacrylamide gel. The spots were analyzed by MALDI-MS. The proteins identified are designated with the accession numbers of the corresponding database.


• In total, 273 different gene products were identified from all gels:

Summary of the 2-D gel electrophoresis data

65 gene products were only detected in the gels carrying total52 in the gels carrying cytosolicremaining proteins were found in both samples

• 45 proteins out of the 62 found in the gels carrying total protein samples45 proteins out of the 62 found in the gels carrying total protein samples were detected in the broad pH range 3–10 gel, 11 in the narrow pH range and nine in both types of gels

• 52 proteins only detected in the gels carrying the cytosolic fraction, except52 proteins only detected in the gels carrying the cytosolic fraction, except for 6 which were found in the broad pH range 3–10 gel, were found in one of the narrow pH range gels only (narrow pH range strips helped to detect 46 proteins not found in the broad range gels)

• Protein distribution was based on the protein identification by mass spectrometry and may not be complete due to:

spot loss during automatic excisionpeptide loss mainly from weak spotspeptide loss mainly from weak spotsspot overlappingsmall protein size

• About 5000 spots were excised from 13 2-D gels, 5 carrying total and 8 carrying cytosolic proteins. The analysis resulted in the identification of about 3000 proteins, which were the products of 273 different genes


Summary of the 2-D gel electrophoresis data


Animals:Male Wistar rats (10–12 weeks, bw: 225±8 g) ( g)Treatment:Bromobenzene (i.p., 5.0 mmol/kg bw)(i.p., 5.0 mmol/kg bw)dissolved in corn oil (40% v/v)Duration of treatment:24 hrs24 hrs

The bromobenzene dose was hepatotoxic, and this was confirmed by the finding of a nearly complete glutathione depletion at 24 hr after bromobenzene administration. The low level of oxidised (GSSG) relative to reduced glutathione (GSH) indicates that the depletion is primarily due to conjugation and to a much lesser extent due tothat the depletion is primarily due to conjugation and to a much lesser extent due to oxidation of glutathione. The bromobenzene administration resulted in on average 7% decrease in body weight after 24 hr.

From: Heijne et al. (2003)

• Liver samples, total RNA (50 g/array experiment)

Gene Expression Profiling

p ( g y p )• cDNA microarrays (3000 genes)• Reference sample:

pooled RNA from liver (~50% w/w), kidneys, lungs, brain, thymus, testes, spleen, heart, and muscle of untreated Wistar rats

• Duplicated microarray/sample• 2-Fold cutoff (p<0.01) relative to the vehicle control:

f f32 genes were found to be significantly upregulated and 17 were repressed following bromobenzene treatment

• 1.5-Fold cutoff (p<0.01) relative to the vehicle control:63 f d t b i ifi tl l t d d 3563 genes were found to be significantly upregulated and 35 genes were repressed following bromobenzene treatment

• Functional groups:Drug metabolismDrug metabolismGlutathione metabolismOxidative stressAcute phase responseProtein synthesisProtein degradationOthers From: Heijne et al. (2003)

Glutathione metabolism:

Oxidative stress:


Acute phase response:


Protein Expression Profiling

• 3 two-dimensional gels were fprepared from each sample

• A reference protein pattern contained 1124 protein spots

• 24 proteins were differentially


• 24 proteins were differentially expressed (BB or Corn oil)

Liver is unique in its capability to regenerate after an injury. Liver regeneration after a 2/3 partial hepatectomy served as a classical modelregeneration after a 2/3 partial hepatectomy served as a classical model and is adopted frequently to study the mechanism of liver regeneration. In the present study, semi‐quantitative analysis of protein expression in mouse liver regeneration following partial hepatectomy was performed using an iTRAQ technique. Proteins from pre‐PHx control livers and livers regenerating for 24, 48 and 72 h were extracted and inspected using 4‐plex g g , p g pisotope labeling, followed by liquid chromatography fractionation, mass spectrometry and statistical differential analysis. A total of 827 proteins were identified in this study. There were 270 proteins for which quantitative information was available at all the time points in both biologically duplicate experiments. Among the 270 proteins, Car3, Mif, dh b b b d lAdh1, Lactb2, Fabp5, Es31, Acaa1b and LOC100044783 were consistently

down‐regulated, and Mat1a, Dnpep, Pabpc1, Apoa4, Oat, Hpx, Hp and Mt1 were up‐regulated by a factor of at least 1.5 from that of the controls at one time point or more. The regulation of each differential protein was also demonstrated by monitoring its time‐dependent expression changes during the regenerating process We believe this is the first report toduring the regenerating process. We believe this is the first report to profile the protein changes in liver regeneration utilizing the iTRAQproteomic technique.

Proteome Res., 5 (7), 1586 -1601, 2006

Systems Toxicology: Integrated Genomic, Proteomic and Metabonomic Analysis of Methapyrilene Induced Hepatotoxicity in the Rat Analysis of Methapyrilene Induced Hepatotoxicity in the Rat

Andrew Craig, James Sidaway, Elaine Holmes, Terry Orton, David Jackson, Rachel Rowlinson, Janice Nickson, Robert Tonge, Ian Wilson, and Jeremy Nicholson

Abstract:

Administration of high doses of the histamine antagonist methapyrilene to rats causes periportal liver necrosis. The mechanism of toxicity is ill-defined and here we have utilized an integrated systems approach to understanding the toxic mechanisms by combining proteomics, metabonomics by 1H NMR spectroscopy and genomics by microarray gene expression profiling. Male rats were dosed with methapyrilene for 3 days at 150 mg/kg/day, which was sufficient to induce liver necrosis, or a subtoxic dose of 50 mg/kg/day. Urine was collected over 24 h each day, while blood and liver tissues were obtained at 2 h after the final dose The resulting data further define the changes that occur and liver tissues were obtained at 2 h after the final dose. The resulting data further define the changes that occur in signal transduction and metabolic pathways during methapyrilene hepatotoxicity, revealing modification of expression levels of genes and proteins associated with oxidative stress and a change in energy usage that is reflected in both gene/protein expression patterns and metabolites. The difficulties of combining and interpreting multi-omic data are considered.

Vehicle 10 mg/kg, 7 days

Methapyrilene-induced liver injury in the rat

100 mg/kg 7 days100 mg/kg 7 days 100 mg/kg, 7 days100 mg/kg, 7 days

Hamadeh et al 2002 Tox Path

“Systems Toxicology: Integrated Genomic, Proteomic and Metabonomic Analysis of Methapyrilene Induced Hepatotoxicity in the Rat”

Proteins altered and identified between control and methapyrilene dosed groups. Proteins are numbered

Ex where elevated and Rx where reduced.

Average standard 1H NMR spectra of liver from each treatment group. This figure shows clearly dose related elevationsand composition changes in fatty acid species…


• Our aim was to determine the impact of drug toxicity on hepatic metabolic pathways and also ascertain whether a lti i t bi l h ld lt i i d d t di f th h i f h t t i it f thmultiomic systems biology approach would result in improved understanding of the mechanism of hepatotoxicity of the

drug

• The combination of information from gene, protein and metabolite levels provides an integrated picture of the response to methapyrilene-induced hepatotoxicity with mutually supporting and mutually validating evidence arising from each biomolecular level. As expected there were several instances where genes and proteins, either encoded by the same gene or by other genes within the same pathway, were both co regulated by methapyrilene toxicity, and sometimes this was in concert with an associated metabolic product

However:

Strategy of parallel omic data sets: It should be noted that alterations in expression of genes or enzyme levels and modification of protein forms while suggesting a potential target of toxic effects do not imply that function or activitymodification of protein forms, while suggesting a potential target of toxic effects, do not imply that function or activity must be altered… Alterations to metabolic profiles reflect function and so may serve to aid interpretation of corresponding gene expression and proteomic analyses… Furthermore, as metabolites unlike genes do not suffer the problem of orthology, observed metabolic effects are likely to be highly conserved between species and integrated systems approaches applied to two species may be one framework within which to reconcile and understand the similarities and differences in genetic wiring of common biological processes between different species.differences in genetic wiring of common biological processes between different species.

Issue of experimental design: …looking at time points where toxicity is already well developed mitigates against obtaining a clear understanding of the temporal dynamics of the mechanism, especially as changes at the gene, protein and metabolite level may proceed at different rates and on different time scales. As such we might expect highly non linear relationships between the concentrations of various species at the different levels of biomolecular organization…

Issue of molecular resolution: …we detected 100s of gene expression changes compared to the relatively small number of changes detected by the other two technologies. It may thus be likely that insufficient detail was obtained at each biomolecular level to elaborate fully on mechanism of methapyrilene toxicity…

Statistical difficulties: Since each data type usually requires tailored preprocessing (normalization, transformation, scaling, etc.) combining multiple data sets presents a significant analytical challenge. Here, we have performed a separate analysis at the gene, protein, and metabolite level and integrated the knowledge gained from each data set to uncover pathways which responded to the methapyrilene-induced toxicity.

pt iproteomics - university of north carolina at chapel hill · 2010-11-17 · the web for peptides...

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