Protein microarrays

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Protein microarrays

• Martyna Franczuk

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Adriana Lagraulet, Journal of the Association for
Laboratory Automation (2010)
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Protein arrays

• 25 000 genes and 1 milion proteins!
• Enable functional protein analysis (in contrast
  with traditional protein profiling).

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Marina Cretich , Francesco Damin, Giovanna Pirri,
Marcella Chiari, Biomolecular Engineering (2006)
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Three types of protein microarrays

• (1) analytical protein microarrays
• (2) functional microarrays
• (3) reverse phase microarrays

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Analytical protein microarrays

• Constructed by arrying antibodies, aptamers,
  affibodies or nonfolded antigens on the glass
  slide.
• Based on the sandwich ELISA methodology.
• Used to profiling protein mixtures
• binding affinities,
• specificities,
• protein expression levels.
• Most common antobody microarrays
• Monitoring differential expression profiles and
  clinical diagnostics

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Functional microarrays

• Contain full-length functional proteins or
  protein domains attached to porous polymer
  matrix.
• Used for studying protein interactions with other
  macromolecules or small molecules and for testing
  entire proteome biochemical activities.

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Reverse phase microarrays

• Cell lysate or plasma are arrayed onto a glass or
  nitrecellulose slide.
• Probing with antibodies against the protein of
  interest.
• evaluation of protein expression levels,
  determination of the presence of altered proteins
  and dysfunctional protein pathways

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Claudius Mueller, Lance A. Liotta, Virginia
Espina, Molecular Oncology (2010)
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Protein chip preparation

• Obtaining proteins from protein libraries
• immobilizing proteins onto a treated microscope
  slide using a contact spotter or a non-contact
  microarrayer

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Protein chip preparation
David A. Hall, Jason Ptacek, and Michael Snyder,
Mechanisms of Ageing and Development (2007)
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Protein chip preparation

• Random attachment and passive adsorbtion glass
  surface with nitrocellulose, gel pads, or
  poly-L-lysine
• Random attachment amines, aldehyde- and
  epoxy-derivatized glass surfaces
• Uniform orientation affinity tag surfaces
• Aqueous environment epoxy-traeted microwells
• Direct protein production on chips

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Detection of moleculeprotein interactions

• Label-based
• covalent conjugation of N-hydroxysuccinimide
  ester-linked fluorescent dyes to primary amines
  of the protein N-terminus or by thiol-reactive
  dyes to cysteine residues
• sandwich ELISA detection method
• tyramide signal amplification (TSA)

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http//hmds.org.uk/histology.html
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Detection of moleculeprotein interactions

• Problem possibility that the label itself may
  interfere with the probes ability to interact
  with the target protein gt
• Label-free
• surface plasmon resonance (SPR),
• carbon nanotubes, carbon nanowires, and
  microelectromechanical systems cantilevers

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Applications

• Discovery of drug targets.
• Sera screening for the presence of autoantibodies
  or viral specific antibodies.
• Cancer and pathogen diagnostic.

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Protein array technology to detect HER2
(erbB-2)-inducedcytokine signature in breast
cancer

• Alejandro Vazquez-martina, Ramon Colomera,
  Javier A. Menendeza
• European Journal of Cancer 4 3 ( 2 0 0 7 )

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Introduction

• HER2 overexpression and breast cancer
• Human Cytokine Array III
• Media from MCF-7 breast cancer cell lines and
  plasma from patients
• Interleukin-8 nad Growth-related oncogene (GRO)
  may represent a ptahway involved in tumor
  progression

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(a) Template showing the location of
cytokine antibodies spotted onto the RayBioTM
Human Cytokine Array III.
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Pharmacological inhibition of HER2-drivencellular
signalling knock-down IL-8 and GRO expression
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Detection of cytokine expression from metastatic
breast cancer (MBC) patients sera.
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High Diagnostic Accuracy of AntigenMicroarray
for Sensitive Detection ofHepatitis C Virus
Infection

• Jung-ah Kwon, Hyeseon Lee, Kap No Lee,
• Kwangchun Chae, Seram Lee, Dong-ki Lee, and
• Soyoun Kim
• Clinical Chemistry 54 (2008)

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Introduction

• Confirmatory test for blood serodiagnosis
• 96-well antigen microarray on sol-gel derived
  material
• Patients sera samples previously tested for HCV
  by ELISA
• Assay time and conditions compatible with
  conventional ELISA diagnostic assay

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Literature

• Adriana Lagraulet, Current Clinical and
  Pharmaceutical Applications of Microarrays From
  Disease Biomarkers Discovery to Automated
  Diagnostics in Journal of the Association for
  Laboratory Automation 15 (2010), 405-413.
• Marina Cretich , Francesco Damin, Giovanna Pirri,
  Marcella Chiari, Protein and peptide arrays
  Recent trends and new directions in
  Biomolecular Engineering 23 (2006), 7788.
• David A. Hall, Jason Ptacek, and Michael Snyder,
  Protein microarray technology in Mechanisms of
  Ageing and Development 128 (2007), 161167.
• Claudius Mueller, Lance A. Liotta, Virginia
  Espina, Reverse phase protein microarrays advance
  to use in clinical trials in Molecular
  Oncology 4 (2010), 461-481.
• Presented original articles