Calibration%20and%20Normalization%20of%20Protein%20Microarray%20Data

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Calibration and NormalizationofProtein
Microarray Data

• Charlene Liang1, Virginia Espina2, Julia
  Wulfkuhle2,
• Emanuel F. Petricoin3 III and Lance A. Liotta2,
  Yuexia Li1, Minzi Ruan1,
•  
• 1VigeneTech Inc,
• 2National Cancer Institute, Center for Cancer
  Research, Laboratory of Pathology,
• FDA-NCI Clinical Proteomics Group,
• 3Food and Drug Administration, Office of Cellular
  and Gene Therapy, Center for Biologic Evaluation
  and Research,
• FDA-NCI Clinical Proteomics Group, Bethesda, MD
• Correspondence cliang_at_vigenetech.com

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Abstract

• Reverse Phase Protein microarrays are a
  promising technology for characterization of
  cellular protein signaling networks. This
  platform has been shown to have high sensitivity
  and good reproducibility when used with validated
  antibodies. There has been a need in clinical
  research to quantify individual analytes across
  patient samples, as well as comparison of
  analytes before, during and after treatment. In
  addition, there has been a need to develop a
  method of normalization and calibration for the
  microarray. We have developed a method of
  normalization based on total protein per
  microarray spot by using a total protein stain on
  the microarray. This allows normalization of
  each spot to a known analyte, maximizing
  reproducibility. We also developed a reference
  lysate of known composition for quantification of
  spots based on a common unit, which we termed the
  reference standard unit. This reference lysate
  serves as a standard for compensation of
  spot-spot and day-to-day variation.
• Bioinformatic software capable of
  incorporating the normalization and calibration
  data is required for high throughput data
  analysis. We used MicroVigene software to
  quantify each analyte on the protein microarray,
  incorporating the reference lysate and total
  protein/spot data. A variety of automated curve
  fitting approaches are used to meet the
  coefficient of variation required for clinical
  trial research.

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Reverse Phase Protein Array
Coupling Laser Capture Microdissection With High
Throughput Protein Arrays
Patient biopsy tissue cells are
microdissected 30,000 cells 100 arrays
Each patient sample is arrayed in a miniature
dilution curve Always in linear dynamic range
of any antibody/ analyte pair
Arrays probed with labeled amplified
antibody e.g. Ovarian cancer progression From
one patient probed with Phospho-ERK antibody
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Application Areas of the Technology

• Reserve Phase Protein Microarrays are applied to
• Clinical Research utilized in clinical trials
  for assessing response to therapy and
  demonstrating protein molecular changes to
  therapy.
• Disease Prognostics - utilized for determining
  which patient is likely to respond to a given
  therapy.
• Personalized drug treatment monitoring response
  to therapy before, during and after treatment.

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Experimental Design
Experimental Design
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Arrayer and Stainer used For Reverse-Phase
Protein Arrays
DakoCytomation Autostainer for protein
detection/signal development
GMS 417 pin and ring arrayer
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Reverse Phase Protein Microarray Methodology

• Tissue processing and microdissection
• 8.0 µm frozen sections of ovarian cancer tissues
  were placed on uncoated glass slides and stored
  at -80ºC prior to use. Ovarian tumor epithelial
  cells or other relevant cell populations were
  microdissected with a Pixcell II Laser Capture
  Microdissection system (Arturus). Approximately
  5,000 LCM shots (20,000-25,000 cells) were
  microdissected for each case and stored on
  microdissection caps at -80ºC until lysed.
• Cell lysis and cellular lysate arraying
• Microdissected cells were lysed directly from
  the microdissection caps into 50 µL of lysis
  buffer containing a 11 mixture of 2x
  Tris-Glycine SDS sample buffer (Invitrogen Life
  Technologies) and Tissue Protein Extraction
  Reagent (Pierce) plus 2.5 ß-mercaptoethanol for
  30 min at 75ºC. Positive control samples
  included A431 control and A431EGF lysates (BD
  Pharmingen) at 1.0 mg/mL. Reference standard
  peptides specific for the pAkt and pERK
  antibodies (Cell Signaling Technology) were
  diluted in lysis buffer to 1.0 µg/mL.
  Immediately prior to arraying, lysates were
  loaded into a 384-well plate and serially diluted
  with lysis buffer into a 5-point dilution curve
  (ovarian samples and A431 controls) ranging from
  undiluted-116 or 12-point dilution curve
  (reference standard peptides) ranging from
  undiluted-116. Approximately 60 nL of each
  sample was spotted onto nitrocellulose-coated
  glass slides (Schleicher and Schuell Bioscience)
  with a GMS 417 microarrayer (Affymetrix). Slides
  were stored dessicated at -20ºC. For estimation
  of total protein amounts, selected arrays were
  stained with Sypro Ruby Protein Blot Stain
  (Molecular Probes) according to the
  manufacturers instructions and visualized on a
  Fluorchem imaging system (Alpha Innotech). One
  day prior to antibody staining, the lysate arrays
  were treated with Reblot antibody stripping
  solution (Chemicon) for 15 min at room
  temperature, washed 2 x 5 min in PBS, and then
  incubated overnight in blocking solution (1g
  I-block (Tropix), 0.1 Tween-20 in 500 mL PBS) at
  4ºC with constant rocking.
• Protein microarray staining 
• Blocked arrays were stained with antibodies on
  an automated slide stainer (Dako Cytomation)
  using the Catalyzed Signal Amplification System
  kit according to the manufacturers
  recommendation (CSA Dako Cytomation). Briefly,
  endogenous biotin was blocked for 10 min using
  the biotin blocking kit, followed by application
  of protein block for 5 min primary antibodies
  were diluted in antibody diluent and incubated on
  slides for 30 min and biotinylated secondary
  antibodies were incubated for 15 min. Signal
  amplification involved incubation with a
  streptavidin-biotin-peroxidase complex provided
  in the CSA kit for 15 min, and amplification
  reagent, (biotinyl-tyramide/hydrogen peroxide,
  streptavidin-peroxidase) for 15 min each.
  Development was completed using
  diaminobenzadine/hydrogen peroxide as the
  chromogen/substrate. Slides were allowed to air
  dry following development.
• Primary antibodies used in these studies were
  Akt 1100 (Cell Signaling Technology) phosphoAkt
  S473 150 (Cell Signaling Technology) phosphoAkt
  T308 150 (Cell Signaling Technology)
  extracellular signal-regulated kinase (ERK) 1/2
  1200 (Cell Signaling Technology) phosphoERK1/2
  T202/Y204 11000 (Cell Signaling
  Technology Secondary antibody and dilution used
  was biotinylated goat anti-rabbit IgG (HL) at a
  15000 dilution (Vector Laboratories).

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Printing Arrays
Single Pad Format
Sector Format
Multiple samples may be printed on a single slide
in the single pad format. Alternatively, one
sample can be printed in six separate sectors.
Each slide contains patient samples, standards
and controls.
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Normalization Using Total Protein

• Why normalization?
• It is essential to account for differences in
  total protein concentration between each sample
  so that antibody staining between each tissue
  sample on the array can be compared directly.
• How do we do it?
• One slide/printing is stained for total protein
  using a total protein stain such as Sypro Ruby
  blot stain or a colloidal gold stain. For
  estimation of total protein amounts, selected
  arrays were stained with Sypro Ruby Protein Blot
  Stain (Molecular Probes) according to the
  manufacturers instructions and visualized on a
  Fluorchem imaging system (Alpha Innotech).
• Reproducibility
• Normalization is based on the total protein per
  microarray spot. Normalized intensity values are
  calculated by dividing the measured intensity
  value of the antibody by the corresponding
  measured intensity value of the total protein.
  This allows normalization of sample to a known
  analyte, maximizing reproducibility.

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Negative Control Slides

• Why negative slides?
• Serve as controls for non-specific binding of
  the secondary antibody to the array
• How is it done?
• Arrays are probed with the labeled secondary
  antibody (biotinylated anti-rabbit or anti-mouse
  IgG) in the absence of the primary antibody
  against the analyte of interest and processed as
  all other slides in the experiment

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Cross Sample Calibration using Reference Standard

• The reference standard is a pool of peptides.
  This pool is comprised of the peptides used as
  the immugen to produce the primary antibody.
• The reference standard dilution curve is
  printed on each microarray slide. MicroVigene
  software automatically finds RSU measurements for
  all samples through curve fitting and other
  mapping algorithms.

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Image Quantification and Data Analysis Using
MicroVigene

• Quality
• Reproducibility
• Automation
• Dust and Contamination Control
• Performance for High Throughput

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Quality Images Actual Spots Boundary, Regional
Background Correction
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Reproduce Same Results

• Same sample stained two months later produces
  over 90 correlation

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Automation - One Mouse Click Operation
Sample Control Images
Image Analysis Grid Spot Quantification
Reverse Phase Protein Microarray
Measurement Linear Range RSU Index Best Linear
Point
Dilution Curve fitting Linear non_linear Dynamic
switch
Background correction Regional Negative Ctrl
Replicates Spot level Curve level Protein Level
Normalization Internal Total Protein
Quality Control Outliers Bad Curves
Output Measurements Error bars Curve fitting
quality flag
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Find the Linear Range through Robust Curve Fitting
Bad spot
Line represent linear range
Nonlinear 4-parameter fitting
Automatically remove the spot
linear fitting
MicroVigene starts with a nonlinear logistic
model. If the number of reliable points outliers
is less than five after removing, the program
will automatically switch to a linear model. The
end results are the optimal fitting of either
linear or nonlinear model.
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Choosing the Right Linear Range

• Smallest error and largest linear range, graph
  with outliers

51 linear range
Nonlinear regression fitting
Not chosen Small slop or shorter linear range
Chosen Linear range line Larger slop longest
linear range
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Best Linear Point Measurements
Y
Taking Y intensity measurements at X0-average
provides the least error due to extrapolation and
offers means for sample comparison.
X0-avg
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Reference Standard Unit Mapping
First we find the best intensity measurement Y0,
then find the point on the rsu dilution curve,
whether linear range line or the actual curve
fitting line, the corresponding x is the sample
concentration (or dilution) measured in the
reference standard unit.
Y0(100)
rsu
Y0(100)
Sample
x
rsu 100 ex
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Measurements for Control Types

• A431 and A431EGF lysates are printed on
  each slide as process controls. Expected results
  are relative elevation of pERK and pAKT in the
  A431EGF lysate as compared to the A431 lysate.

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Results Relative Signal Intensity
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Relative Signal Intensity by Disease Stage
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Using Reference Standard Unit (RSU)
It is important to make measurements in the
linear range of protein concentration. In order
to be able to compare results across samples and
periods, we have introduced RSU measurements. We
are showing here that correlation between Y0 and
RSU is proportional.
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Results Reference Standard Unit (RSU)
RSU and Y0 give close relative measurements
between antibodies.
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Conclusion

• The detection of changes in the activity of
  various signaling pathways in
• normal and tumor tissue in a patient is essential
  for understanding disease
• progression, appropriate treatment selection, and
  monitoring treatment
• efficacy. Reverse Phase Protein Microarray
  technology provides a means to detect, in a
  highly multiplex way, these changes. With the
  standardization on the total protein
  normalization and the incorporation of a
  reference standard, Reverse Phase Protein
  Microarrays make data comparison among different
  studies and clinical trials possible.
• This analysis is performed using MicroVigene
  software employing
• advanced algorithms for image analysis and novel
  data analysis processes for high quality, highly
  reproducible, end-to-end Reverse Phase Protein
• Microarray analysis solutions. MicroVigene also
  automates the
• Calibration, normalization and background
  correction analysis steps of the method.

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ABOUT US

• The goal of the FDA-NCI Clinical Proteomics
  Program is to invent and apply proteomics
  technology to patient care. New proteomics
  research technology is now being used for
  clinical studies ranging from cancer to
  cardiovascular disease and organ transplant.
  Researchers within the program are searching for
  proteins in the blood, urine, and diseased tissue
  that can be used as early biomarkers of disease,
  predict response to therapy, or the likelihood of
  relapse after treatment, or serve as new targets
  for therapy itself.
• About VigeneTech, Inc. VigeneTech provides novel
  scientific software, customized solutions and
  online services in the areas of image analysis,
  automation, and instrumentation. VigeneTechs
  MicroVigeneTM for microarray image analysis
  delivers 100 reproducible, operator independent
  results it is robust to handle various shifted
  and noisy images and supports unattended batch
  process. For more information about VigeneTech,
  please visit http//www.vigenetech.com.