Advances in urinary proteome analysis and biomarker discovery
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Advances in urinary proteome analysis and biomarker discovery. Joost Schanstra Inserm U858, I2MR Toulouse, France. B L O O D. U R I N E. - Serum versus plasma - Proteolysis. - Simple pre-analytic handling - Stable. Reduction of sample variability. Biomarkers in biofluids.

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Advances in urinary proteome analysis and biomarker discovery

Advances in urinary proteome analysis and biomarker discovery

Joost Schanstra

Inserm U858, I2MR

Toulouse, France


Advances in urinary proteome analysis and biomarker discovery

B L O O D

U R I N E

- Serum versus plasma

- Proteolysis

- Simple pre-analytic handling

- Stable

Reduction of sample variability

Biomarkers in biofluids

Petricoin et al., Use of proteomic patterns in serum to identify ovarian cancer.

Lancet. 2002

  • Irreproducible: - improper mass calibration,

    • technical flaws,

  • - improper execution of the experimental protocol


  • Advances in urinary proteome analysis and biomarker discovery

    ~30%

    Urine – pool for biomarkers of other diseases ?

    Urine – pool of biomarkers

    Sources of urinary proteins

    1) Filtration and secretion of plasma proteins.

    2) Secretion by various renal segments.

    3) Proteolytic degradation products of extracellular matrix

    4) Secretion by the urinary tract.

    5) Dead cells.

    ~70% of the urinary proteins/peptides originate from the kidney and urinary tract

    ~70%

    Kidney “health” status


    Advances in urinary proteome analysis and biomarker discovery

    Advances in urinary proteome analysis and biomarker discovery

    Advances in techniques


    Advances in urinary proteome analysis and biomarker discovery

    250

    150

    100

    75

    50

    37

    25

    20

    15

    Reduce complexity

    A one-dimensional view of the urinary protein content

    • - Dynamic range

    • A few proteins make up the majority

    Courtesy: C. Lacroix, Toulouse


    Advances in urinary proteome analysis and biomarker discovery

    Reduce complexity

    Fractionation

    Mass spectrometry

    Sample

    2D-PAGE

    SELDI

    Capillary electrophoresis


    Advances in urinary proteome analysis and biomarker discovery

    250

    150

    100

    75

    50

    37

    25

    20

    15

    Two-dimensional gel electrophoresis

    albumin

    Thongboonkerd, 2002

    Mass (kDa)

    microglobulin

    Ig kappa chain

    acidic

    basic


    Advances in urinary proteome analysis and biomarker discovery

    SELDI-MS

    (surface enhanced laser desorption/ionisation-mass spectrometry)

    100

    250

    Mass (kDa)

    150

    100

    75

    50

    37

    25

    20

    15

    3


    Advances in urinary proteome analysis and biomarker discovery

    250

    150

    100

    75

    50

    37

    25

    20

    15

    Capillary electrophoresis coupled to mass-spectrometry

    Coon, in press.

    • - 5000 frequently

    • occurring peptides

    • collagen fragments !!

    Mass (kDa)

    Migration on CE (time)


    Advances in urinary proteome analysis and biomarker discovery

    Three techniques suitable for the analysis of the urinary proteome

    SELDI

    2D-PAGE

    CE-MS

    Medium throughput

    High throughput

    More amendable for use in the clinic


    Advances in urinary proteome analysis and biomarker discovery

    Multiple biomarkers or “panels” of biomarkers

    proper statistics !!

    Advances in urinary proteome analysis and biomarker discovery

    Advances in concepts: panels rather than individual markers

    • Individual markers:

    • Lack of specificity (PSA, microalbuminuria…)

    • Sensitive to inter- and intra-individual variations


    Advances in urinary proteome analysis and biomarker discovery

    Advances in urinary proteome analysis and biomarker discovery

    Advances in concepts: standards


    Advances in urinary proteome analysis and biomarker discovery

    Biochemists

    2. Select appropriate patient groups

    /clinical data/samples/proteomic analysis

    3. Training population

    Statisticians/

    bioinformatics

    extract biomarkers/

    proper statistics!!

    Proteomics

    core platforms

    5b. Identify

    5a. Large scale validation

    pathophysiology

    clinic

    A standard flow scheme for clinical proteomics

    • Define a clear clinical question

    • - surgery or not ?

    • - graft rejection or not ?

    Clinicians

    4. Validate in separate blinded population

    Mischak, Apweiler, Banks, Conaway, Coon, Dominiczak, Ehrich, Fliser, Hermjakob, Hochstrasser, Jankowski, Julian, Kolch, Massy, Neusuess, Novak, Peter, Rossing, Schanstra, Semmes, Theodorescu, Thongboonkerd, Weissinger, Van Eyk, Yamamoto Proteomics Clin. Appl. 1: 148 (2007).


    Advances in urinary proteome analysis and biomarker discovery

    Advances in urinary proteome analysis and biomarker discovery

    Age matching

    Biomarkers selected for this age range

    old

    young

    ?

    ?

    old

    young


    Advances in urinary proteome analysis and biomarker discovery

    Secretion of 325 out of 5000 urinary peptides is modified during aging

    Advances in urinary proteome analysis and biomarker discovery

    Age matching

    324 individuals: 2-73 year

    Analysis of the urinary proteome by CE-MS of 324 individuals


    Advances in urinary proteome analysis and biomarker discovery

    Age match !!!!!

    Renal aging = CKD ?

    Zurbig et al., submitted


    Advances in urinary proteome analysis and biomarker discovery

    Outliers !!

    age

    19-30 31-40 41-50 51-73

    age markers

    y

    x

    ??

    Zurbig et al., submitted


    Advances in urinary proteome analysis and biomarker discovery

    Advances in urinary proteome analysis and biomarker discovery

    Advances: examples

    • Renal allograft rejection

    • Ureteropelvic junction obstruction

    • 3. Bladder Cancer (BCa)


    Advances in urinary proteome analysis and biomarker discovery

    Classification:

    healthy

    stable transplants

    acute rejection

    Random

    forrest

    Acute rejection versus stable:

    - 4 markers (sens 90,5%, spec 83,3%)

    Prediction of acute rejection in renal allografts

    Year 2004

    Training

    20 healthy

    22 stable transplants

    23 acute rejection

    SELDI

    O’Riordan et al., 2004. JASN 15: 3240


    Advances in urinary proteome analysis and biomarker discovery

    - -defensin-1

    - -1-antichymotrypsin

    Validation of -defensin-1

    in immunoassay

    AUC 0.749

    Loss of sensitivity !

    O’Riordan et al., 2007. Am J Transpl. 7:930

    Prediction of acute rejection in renal allografts

    Year 2007

    MS-based

    Immunoassay based

    Validation (n=45)

    Two markers

    AUC 0.912

    Stable versus acute

    Sensitivity 91.3 %

    Specificity 77.3 %


    Advances in urinary proteome analysis and biomarker discovery

    Obstructive nephropathy

    scintigraphies

    Spontaneous

    resolution

    Spontaneous

    resolution

    Intermediate

    obstruction

    ?

    pyeloplasty

    pyeloplasty

    Ureteropelvic junction (UPJ)

    obstruction

    birth

    1 to 2 years

    Objective:identify early urinary biomarkers

    indicative for surgery


    Advances in urinary proteome analysis and biomarker discovery

    Extraction of group-specific

    markers

    Set of specific biomarkers

    separating different groups:

    Classification

    Support vector machines

    Training

    Patient groups:

    Individual CE-MS

    Profiles:

    Reference (compliled)

    Group profile:

    Healthy controls

    (n=13)

    Sp. Resolution

    (n=19)

    Pyeloplasty

    (n=19)

    1

    1

    1

    19

    19

    13


    Advances in urinary proteome analysis and biomarker discovery

    9 months: 34 out of 36 patients correctly predicted

    15 months: 35 out of 36 patients correctly predicted

    Decramer et al., 2006. Nat Med. 12: 398.

    Small scale (n=36) prospective blinded validation

    Biomarkers obtained during « training »

    Intermediate

    Obstruction

    (n=36)

    CE-MS based

    prediction

    Spontaneous

    resolution

    prediction

    Clinical outcome

    pyeloplasty

    1 month

    1 to 2 years

    birth

    Large scale multicenter validation of markers started in 2008: 358 patients


    Advances in urinary proteome analysis and biomarker discovery

    • Reoccurrence

    • cystoscopy (invasive)

    • urine cytology (low-sensitivity)

    Urinary proteomics to search for biomarkers of BCa

    Bladder cancer

    Transitional cell carcinomas

    80% superficial (Tis, Ta, T1)


    Advances in urinary proteome analysis and biomarker discovery

    Transitional cell carcinoma detection: Training

    group

    Number of patients

    Training set

    Bladder cancer

    46

    controls

    73

    Confounding disease

    various Renal diseases

    281

    Prostate cancer

    21

    Renal cancer

    24


    Advances in urinary proteome analysis and biomarker discovery

    Training set

    Controls + confounding

    Bladder cancer

    22 biomarkers


    Advances in urinary proteome analysis and biomarker discovery

    Suitable biomarkers in the presence of confounding diseases

    Clinical setting

    Transitional cell carcinoma detection: Validation

    Controls + confounding diseases

    Bladder cancer (n=32)

    versus

    Controls (n=12)

    Various renal diseases (n=131)

    Nephrolithiasis (n=7)

    32/32 Bladder cancer Sensitivity 100 %

    12/12 ControlsSpecificity 100 %

    124/131 Various renal diseases Specificity 95 %

    6/7 Nephrolithiasis Specificity 86 %

    Theodorescu et al., Lancet Oncol. 7:230-240 (2006)


    Advances in urinary proteome analysis and biomarker discovery

    Advances in urinary proteome analysis and biomarker discovery

    Advances: examples

    Urine as a source of biomarkers for non-urogenital diseases?

    Coronary artery disease


    Advances in urinary proteome analysis and biomarker discovery

    Urinary proteomics for biomarkers of Coronary artery disease

    Study Setup

    Training set

    CAD (Glasgow)n = 30

    Controls(Glasgow)n = 20

    Additional controls:

    Ramipril (medication controls)n = 18

    Hannover (center specific biais)n = 232

    Validation set

    CADn = 47

    Controlsn = 12


    Advances in urinary proteome analysis and biomarker discovery

    upregulated in comparison

    Downregulated in comparison

    Training:15 biomarkers differentiate between CAD and non-CAD controls

    patients with CAD

    non-CAD controls


    Advances in urinary proteome analysis and biomarker discovery

    Urinary markers for disease

    from more distant organs

    Low molecular weight proteins:

    glomerular filtration

    Validation of urinary CAD biomarkers

    46/47 CADSensitivity 98%

    10/12 controlsSpecificity 83%

    Zimmerli et al., 2008 Mol. Cell. Proteomics 7, 290-298


    Advances in urinary proteome analysis and biomarker discovery

    852.476

    995.435

    100

    y9

    b10

    882.357

    90

    b9

    80

    tandem

    mass-spectrometry

    1587.759

    b16

    70

    60

    1715.817

    858.408

    b18

    50

    b18 ++

    Intensity

    40

    794.380

    30

    b16 ++

    Sequence identification

    1181.252

    20

    755.422

    y12

    y8

    609.256

    1318.578

    1092.506

    b13

    b6

    10

    b11

    0

    600

    800

    1000

    1200

    1400

    1600

    m/z

    Advances in urinary proteome analysis and biomarker discovery

    From biomarkers to pathophysiology?


    Advances in urinary proteome analysis and biomarker discovery

    Advances in urinary proteome analysis and biomarker discovery

    From biomarkers to pathophysiology?

    • Biomarkers:

    • Abundant plasma proteins and fragments.

    • (albumin, beta2-macroglobulin, alpha1-antitrypsin etc…).

    • Abundant (structural) kidney proteins and fragments.

    • (collagens, uromodullin).

    Not highly informative


    Advances in urinary proteome analysis and biomarker discovery

    Advances in urinary proteome analysis and biomarker discovery

    Chasing low abundance proteins

    A few proteins and their fragments make up the major part

    of the urinary proteome

    Although we fractionate with proteomics these abundant

    proteins still “hide” the less abundant ones


    Advances in urinary proteome analysis and biomarker discovery

    Advances in urinary proteome analysis and biomarker discovery

    Chasing low abundance proteins

    Immunodepletion of

    high abundance proteins

    Human plasma

    - Depletion of human serum albumin

    815 other protein species

    +

    +

    2091 other protein species

    - Depletion of IgGs

    Shen et al., 2005, Proteomics 5: 4034


    Advances in urinary proteome analysis and biomarker discovery

    before after

    Advances in urinary proteome analysis and biomarker discovery

    Chasing low abundance proteins

    LARGE

    DYNAMIC

    RANGE

    REDUCED

    DYNAMIC

    RANGE

    +

    Ligand peptide library

    (millions of different ligands)

    Flow-Through:

    Highly abundant proteins

    Albumin

    IgGs


    Advances in urinary proteome analysis and biomarker discovery

    Advances in urinary proteome analysis and biomarker discovery

    Conclusion

    * Urine has emerged as a stable «  reservoir » of biomarkers for both urogenital and non-urogenital diseases.

    * The variability in single biomarkers is counteracted by patterns that tolerate instability and inconsistency of individual polypeptides/biomarkers

    * First studies using adequate statistics and validation have been published.

    Large scale validation

    * Studies including confounding diseases are being published.

    One step closer to use in the clinic

    * The contribution of proteomics to the understanding of pathophysiology is still limited


    Advances in urinary proteome analysis and biomarker discovery

    Thank you


    Advances in urinary proteome analysis and biomarker discovery

    22/24

    Beyond urine

    Bilateral developmental

    nephropathy

    Post-natal renal function ?

    Presence of specific peptides/proteins

    in amniotic fluid

    Responsible for the modification

    of post-natal renal function

    Early renal lesions invisible

    by sonography

    Clinical question: can we define AF biomarkers predicting post-natal renal function?


    Advances in urinary proteome analysis and biomarker discovery

    842 non-redundant proteins

    1000-1200 peptides/sample

    2D-PAGE

    LC-MS

    CE-MS

    SELDI

    Identification of

    intra-amniotic inflammation

    (n=169, blinded, high spec/sens)

    Amniotic fluid is great source of biomarkers !!

    Buhimschi et al., 2007,

    PLoS Med 4(1): 84-94.

    Beyond urine

    Amniotic fluid !!


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