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  • Proteome Mapping by 2-D HPLC as a Tool for Veterinary Biologic Antigen Identification.

    Timothy J. Barder, Ph.D.

  • 4 Broad Challenges in Proteomics1. No one technique will give all the answers.

    Dynamic Range & Resolution Issues. 104 105 Genes transcribing 105 106 proteins

    2. The proteome is very dynamic. Temporal & environmental influences. Post-translational modifications, P-P interactions.

    3. Many different protein sources. Tissue, Serum/plasma, bio-fluids Inter/Intra-cellular Biology & Extra-cellular Biology

    4. Inclusionary vs. Exclusionary Protein Analysis Whole picture vs. partial picture of expression. Intact protein analysis vs. Peptide mapping.

  • Reproducibility Issues

    Difficult to Automate

    Limited Quantitation; MW & Expression Level

    Difficulties with low MW proteins (

  • 1st Dimension; pI information

    Chromatofocussing using HPLC => Direct Reference to 2-D GelsLiquid pI fractions for direct analysis in second dimension HPLC

    2nd Dimension; MW Information

    RP-HPLC => Orthogonal to pI & analogous to MWMulti-well Fractionation for highly resolved liquid protein fractions

    Is there a liquid phase alternative to 2D Gels?i.e. a 2D Liquid Phase Separation and Mapping System for

    Intact Protein Expression Analysis.

  • Imaging or mapping of protein expression is very useful for future referencing of sample!

    - Not easy to map or compare sample to sample with peptides only.

    - Proteins first imaged & then recovered intact. => PTM info not lost!

    - Limitations of MS: Peptides first? - Why do it the hard way!

    Fractionation of proteins simplifies the complexity problem with no loss of sample information!

    - Intact proteins are easily fractionated in the liquid phase.

    - Can generate useful & reproducible arrays of proteins in 96-well plates => application of other techniques for further analysis.

    2D HPLC Methods: Proteins vs. Peptides?

  • ProteomeLab PF2D System from Beckman CoulterWorks with all types of biological matrices!

    Highly automated & user-friendly.

  • Total Protein Hydrophobicity Profile

  • E. coli O157:H7 Whole Cell Lysate (Strain # 96-0112)

    Protein Hydrophobicity 1D Maps

  • E. coli O157:H7 Whole Cell Lysate (Strain # 96-0112)

    Complex Protein pI Profile

  • pI Fractions taken at fixed pH ranges

    ProteoVue pI/Hydrophobicity 2D Protein Expression Map E. Coli O157:H7 Whole Cell Lysate

    pI

    Retention Tim

    e (min)

    Fraction #

    Hydrophobicity Profile for

    Proteins in pI range = 5.8 6.1

    * Lane 9

    *

  • Two Different E. coli O157 strains from Penn StatepI range 3.5 7; Hydrophobicity range 10 19 min

    ProteoVue pI/Hydrophobicity 2D maps

    E. coli O157:H7 E. coli O157:H32

  • E. coli O157:H7 E. coli O157:H32DeltaVue Difference Map

    All pI Fractions

    DeltaVue images Expression level changes as well as Pattern differences

  • E. coli O157:H7 E. coli O157:H32DeltaVue Difference Map

    pI 6.1-6.4

    DeltaVue Identifies Strain Specific Proteins

  • E. coli O157:H7 E. coli O157:H32

    pI 4.3-4.6

    DeltaVueDifference

    Map

    DeltaVue Measures Protein Expression Level Differences

    H7/H32 = 1.96

    H7/H32 = 0.45

  • Overlay Mode

    Reproducibility of Protein Expression Patterns:Same E. coli O157:H7 sample Run 2 times through ProteoSep

    pI 4.3-4.6

  • Protein Fingerprinting using ProteoVue 2D maps

    E. coli O157:H7E. coli O157:H32 E. coli O157:H45Three different E. coli O157 strains from Penn StatepI range 3.5 7; Hydrophobicity range 10 19 min

  • 2D Protein HPLC and MS:

    Liquid Phase Intact Protein MW Analysis with MS

    Protein ID, Expression level & PTM information!!!

    Insight into total biology of sample or organism from

    gene transcription protein expression.

    Peptide mapping only provides gene based Protein ID information.

    Database MW values often differ from expressed MW values.

    Numerous possible protein modification pathways.

  • Comparison of general E. coli

    strain (88-0477) vs. virulent

    E. coli O157:H7 strains (96-0107

    & 96-0112)

    Zheng, et. al., BioTechniques, 35(6)

    1210-1212 (2003)

  • Green (right) E. coli O157:H7Red (Left) -

    general E. coli

  • **29836/5.443Exu regulon transcriptional regulator - P42608

    **88965/6.822Trimethylamine-N-oxide reductase 2 precursor (TMAO reductase 2) (trimethylamine oxidase 2) -P46923

    **35625/5.230Delta-aminolevulinic acid dehydratase(porphobilinogen synthase) (ALAD) (ALADH) -P15002

    -13534354.71534490/5.834Cysteine synthase A (o-acetylserine sulfhydrylase

    A) (o-acetylserine (thiol)-lyase A) (CSase A) (sulfate starvation-induced protein 5) (SSI5) -P11096

    -2 18151.8218154/5.542Peptidyl-prolyl cis-trans isomerase B (PPIaseB) (rotamase B) - P23869

    Delta MW(Da)

    Actual MW (Da)

    Database MW/pI (Da)

    Sequence Covered (%)

    Protein Name Database Accession #

    Proteins over-expressed in general E. coli

  • +215936.8215935/6.063Unknown protein from 2-D PAGE (spots PR25/LM16/2D_000LR3) - P39177

    -9021801.3221891/5.840Modulator of drug activity B - P40717

    +12937394.2837265/5.926UDP-glucose 4-epimerase (galactowaldenase) (UDP-galactose 4-epimerase) - P09147

    **34455/6.332Curved DNA-binding protein - P36659-411529.1811533/5.840Protein ygiN - P40718

    -8915450.6115540/5.464DNA-binding protein H-NS (histone-like protein HLP-II) (protein H1) (protein B1) -P08936 M

    -13212652.912785/6.247Protein yfiA - P11285

    -13210105.0610237/4.975DNA-directed RNA polymerase omega chain (transcrip-tase omega chain) (RNA polymerase omega subunit) - P08374

    -10719597.5719704/5.035Inorganic pyrophosphatase (pyrophosphate phospho-hydrolase) (PPase) - P17288

    +2021245.821226/9.725Protoporphyrinogen oxidase (PPO) - P27863 -13219406.7919538/5.342Shikimate kinase I (SKI) - P24167

    014284.514284/5.158Protein yfiD - P33633

    Delta MW[Daltons]

    Actual MW (Da)

    Database MW/pI (Da)

    Sequence Covered (%)

    Protein Name Database Accession #

    Proteins over-expressed in E. coli O157:H7

  • **8525/4.781Hypothetical protein yccJ - P46131

    **18665/5.747Molybdenum cofactor biosynthesis protein B -P30746

    **19416/5.249Autoinducer-2 production protein luxS (AI-2 synthesis protein) - P45578

    -3916880.4316919/6.141Methylglyoxal synthase (MGS) - P37066

    -13218563.48618695/5.764DNA protection during starvation protein -P27430

    -13234915.57835048/5.733

    PTS system, mannose-specific IIAB component (EIIAB-Man) (mannose-permeaseIIAB component) (phospho-transferase enzyme II, AB component) (EIII-Man) - P08186

    -239136476.03538867/5.241Spermidine/putrescine-binding periplasmicprotein precursor (SPBP) - P23861

    Delta MW (Da)

    Actual MW (Da)

    Database MW/pI (Da)

    Sequence Covered (%)

    Protein Name Database Accession #

    Proteins found only in general E. coli

  • Delta MW (Da)

    Actual MW (Da)

    Database MW/pI (Da)

    Sequence Covered (%)

    Protein Name - Database Accession #

    AB**15581/5.438TRAM protein - P71198

    AB-417524.3617528/5.128DNA K suppressor protein -P18274

    AB-34736480.2736827/5.630Ornithine carbamoyltransferasechain F (OTCase-2) - P06960

    BB**47005/6.831Transcription termination factor rho - P03002

    BB-13113110.3613241/5.781HIT-like protein ycfF - P36950

    BB-26826095.4726363/7.925Hypothetical protein yaeB -P28634

    BB-33835195.1535533/6.657Glyceraldehyde 3-phosphate dehydrogenase A (GAPDH-A) -P06977

    BB-13313001.3913134/6.152Protein yifE - P27827

    BB, only present in E. coli O157:H7; AB, present in both equally.

  • 1D Total Protein expression map of Patient (Human) Serum

  • 7.6 mg total protein injected on CF column

    Human Serum Protein pI Profile

  • pI

    Retention Tim

    e (min)

    Fraction #

    Color ProteoVue 2D map of Patient Serum Sample

    Human Serum treated with Lysis Buffer No removal of HSA required!!!

    *

    *

  • 1D Protein Hydrophobicity expression maps of Patient (Human) SerumNormal Patient Sepsis Patient

  • 2D Total Protein expression map of Patient (Human) Serum

    Serum treated with Solubilization Buffer.No removal of HSA required!!!

    Normal Patient Sepsis Patient

  • Imaging softwareZoom-in and renormalization

    for imaging of low expressed proteins

    100 uL of Serum analyzedUV LOD ~ 500 picograms

    UV Dynamic Range >104

  • Biological Samples Analyzed Using This 2D HPLC Concept

    Whole Cell Lysates

    Hepatocytes, Breast Cancer, Colon Cancer, Ovarian Cancer, Mouse embryonic stem cells, Yeast, E. coli, Staph Bacteria,

    Rat Brain Tissue, PBMCs, Flow cytometry samples.

    Protein Fluids

    Secreted Proteins (conditioned media), Sera, Plasma, Amniotic Fluid, Ascites, Urine, Various Lavages, CSF.

    Misc. Protein Samples

    Plant extracts, GMO samples, Meat Product extracts,

    Milk/Cheese Extracts

  • 1st Dimension; pI informationHigh Resolution pI Fractionation using HPLC Chromatofocussing

    Liquid protein pI fractions reference to 2-D PAGE analysis.Broad loading capacity with high protein recovery.

    pI fractions can be stored for future additional analyses.

    2nd Dimension; Mass/Hydrophobicity InformationProtein Hydrophobicity Profiling of pI Fractions:

    Direct liquid phase interface to MS possible.High resolution 1D UV (hydrophobic) or Mass Maps.

    Multi-well Fractionation for highly purified liquid protein fractions:Sequencing, LC/MS, MALDI, Westerns, ELISAs, etc.

    pI/Hydrophobicity : A 2D HPLC alternative to 2D Gels!A fully automated All-Liquid Phase Separation and Mapping

    Method for Intact Protein Expression Analysis.

  • Analytical (MS) Methods

    2D Intact Protein HPLC Concept

    Peptide Mapping

    MALDI, LC/MSn, CE/MS, SELDI

    EDMAN Sequencing

    Protein Expression Arrays

    Protein/Target Function Evaluation

    ELISAs

    Isolation & ID of Important

    Protein Targets/Markers

    from Complex Mixtures

    Bioinformatics

    1-D gels

    1D & 2D Westerns

    2D HPLC High Resolution Fractionation of Intact Proteins

    LC/MS - Intact Protein Analysis

    Molecular Biology Methods

    New Gel-Free Methodologies for Proteomics

  • Protein Discovery Lab

    Drug

    Discovery &

    DevelopmentProduct Developm

    entAp

    plic

    atio

    n De

    velo

    pmen

    t

    Kits, Chemistries & Protocols

    Software & Automation

    ProteomeLab PF 2D Instrument & Software Suite

    ProteomeLabPF 2D Kits

    Beckman Coulter, Inc.Exclusive Licensee of

    ProteoSep & ProteoVueTechnology

  • Acknowledgements

    Prof. David Lubman (U of Michigan)

    Suipeng Zheng (U of Michigan)

    Kim Schneider (U of Maryland)

    Dr. Steve Parus (U of Michigan)

    Ron Doucet (Eprogen)

    Linda Lin (Eprogen)

    Phil DuBois (Eprogen)

    For more info visit www.eprogen.com or www.beckmancoulter.com

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