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Lecture 16. Arrays /High-throughput Analyses. References. G. MacBeath, “Protein microarrays and proteomics”, Nature Gen Supplement,32, 526-532 (2002) H. Zhu and M. Snyder, “Protein chip technology”,Curr. Opin.Chem. Biol. 7,55-63 (2003)

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slide1

Lecture 16

Arrays /High-throughput Analyses

slide2

References

  • G. MacBeath, “Protein microarrays and proteomics”, Nature Gen Supplement,32, 526-532 (2002)
  • H. Zhu and M. Snyder, “Protein chip technology”,Curr. Opin.Chem. Biol. 7,55-63 (2003)
  • P. Braun and J. LaBaer, ‘High throughput protein production for functional proteomics”, Trends Biotechnol. 21,383-388 (2003)
  • J. LaBaer and N. Ramachandran, ‘Protein microarrays as tools for functional proteomics”, Curr. Opin.Chem. Biol.9, 14-19 (2005)
  • M. G. Smith et al. “Global analysis of protein function using protein microarrays”, Mech Aging Develop.126, 171-175 (2005)
  • M. Janzi et al., “Serum Microarrays for Large Scale Screening of Protein Levels”, Mol. Cell. Proteomics 4, 1942-1947 (2005)
  • R. B. Jones et al, “A quantitative protein interaction network for the ErbB receptors using microarrays”, Nature, 439:168-74 (2006)
  • D. Bergsma, S. Chen, J. Buchweitz, R. Gerszten and B. B. Haab, “Antibody-Array Interaction Mapping, a New Method to Detect Protein Complexes Applied to the Discovery and Study of Serum Amyloid P Interactions with Kininogen in Human Plasma” Mol. Cell Prot. 9, 446-456 (2010)
  • D. J. O’Connell et al “Integrated protein array screening and high throughput validation of 70 novel neural calmodulin binding proteins” Mol & Cell Prot. (2010) M900324-MCP200
slide3

Microarrays

  • Minaturized and parallel assays essential for large scale and high-throughput biological analyses
  • In principle, capture molecules are immobilized in a very small area and probed for various activities
  • High signal intensities and optimal signal-to-noise ratios can be achieved under ambient analyte conditions
  • DNA/RNA and protein/peptide are similar in design but ask very different questions
slide5

Protein Microarrays (Chips)

  • Applications: measure antibody-antigen, protein-protein, protein-nucleic acid, protein-lipid, protein-small molecule and enzyme-substrate interactions
slide7

Protein Microarrays (Chips)

  • Applications: measure antibody-antigen, protein-protein, protein-nucleic acid, protein-lipid, protein-small molecule and enzyme-substrate interactions
  • Relevant parameters: surface chemistry, capture molecule attachment, protein labeling and detection methods, high-throughput protein/antibody production and applications to analyze whole proteomes
slide8

Requirements for Protein Arrays

  • Construction of an ordered expression library
  • Methods for purifying a large number of proteins
  • Preparing the array
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Analytical vs. Functional

  • Analytical: high density arrays of antibodies, antibody mimics or other proteins used to measure the presence and concentration of proteins in complex mixtures. Great potential for monitoring protein expression or “protein profiling”
  • Functional: sets of proteins or even whole proteomes are used to measure a wide range of functions and activities
slide17

Antibody Arrays

  • Limitation on obtaining suitable reagents
  • There are alternative reagents but these all have problems in terms of obtaining large amounts of high specificity material
  • Specificity is biggest problem
  • Two Ab assays are preferable but requires there be two Abs with different epitope recognition available for each antigen
slide18

Antibodies (cont)

  • Reverse experiments to detect antibodies against known pathological antigens as diagnostic
  • Allergy test using 94 allergens (not just proteins) to detect patient IgE responses in one-step test
  • Similar assays have been used in autoimmune diseases
slide20

Protein antigens

  • Full-length proteins
  • Synthetic peptides
  • Recombinant protein fragments
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Reverse-phase Protein Arrays

  • Forward phase: bait molecule is normally an Ab or antigen; each spot one type of bait protein
  • In reverse phase, analytes are immobilized; each spot has multiple analytes
  • Allows for many test samples (containing analytes) to be analyzed at once
  • Eg: looking for defciency in IgA responses in immunodeficient patients
slide25

2 types of functional arrays

  • Protein spotting microarrays

Chemical linkage

Peptide fusion tags

  • Self-assembling microarrays

NAPPA

slide29

EGFR Signaling

  • 4 members of erbB family
slide31

EGFR Signaling

  • 4 members of erbB family
  • Probed all the potential Tyr phosphorylation sites using SH2 and PTB domains
  • Human genome: 109 SH2/44 PTB
  • Clones: 106 SH2/41 PTB + 10/3 tandem constructs
  • Prepared by large scale bacterial culture
  • 140/160 were soluble; 19 more obtained after refolding
  • Prepared as 96 well arrays
slide32

EGFR Endodomain Interactions

PM

Kinase

Ras-GAP?

domain

Y730

SHP2?

Y789

c-Sr

c

PI3K?

Y845

(phos)

PLC

Y992

Grb2

Y1068

Y1086

Y1

101

Gab1

Y1

148

SHC

Y1

173

slide33

ErbB phosphorylation sites

  • ErbB1 (EGFR): 12
  • ErbB2: 6
  • ErbB3: 11
  • ErbB4: none reported (but 4 included based on similarity to other family members
  • 17-19 residue peptides (61) prepared as probes in phospho- and non-phospho forms with TAMRA tag (rhodamine)
slide35

Interactions detected

  • 5247 interactions measured (from 61 peptides)
  • 353 (6.7%) had Kd < 2µM (their cutoff)
  • 102/115 SH2 domains and 27/44 PTB domains were ‘active’
slide41

Conclusions

  • By using non-competitive format, revealed high-affinity binding sites for SH2 and PTB domains, many not part of ‘consensus’ sequence profile
  • Recruitment sites on ErbB2 much more promiscuous than on other receptors
  • When only high affinity considered, proteins that bind to EGFR constitute a small subset of those that bind to ErbB3
  • EGFR and ErbB2 become much more promiscuous when their concentration is raised; ErbB3 does not. This likely contributes to high oncogenic potential of EGFR and ErbB2
slide45

Conclusions

  • Array of 37,200 redundant proteins containing an estimated >10,000 unique neural proteins was probed with a tagged calmodulin analyte in the presence of Ca+2
  • 76 proteins identified with KD> 1μM
  • 74 of these targets validated by SPR
  • A microarray of the newly identified targets developed (for further studies)
  • 4 proteins (K+ voltage gate channel, CaM kinase-like vesicle-associated protein, EF-hand domain family member A2 and phosphatidylinositol-4-phosphate 5-

kinase, type I, gamma) with newly identified calmodulin domains were further studied with synthetic peptides and isothermal titration calorimetry