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Protein Microarrays: Michael Snyder

This document discusses protein microarrays, which are high-density arrays containing hundreds to thousands of proteins. There are two main types: antibody microarrays, which use antibodies to profile protein expression, and functional protein microarrays, which screen for protein biochemical activities, interactions, and modifications. The document outlines methods for constructing functional protein microarrays using expression libraries to print entire proteomes. It summarizes several screens performed on a yeast proteome microarray that identified new protein-protein and protein-lipid interactions, kinase substrates, DNA-binding proteins, and drug targets. The document calls for establishing community standards around data reporting and repositories to facilitate data sharing and reproducibility.

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180 views44 pages

Protein Microarrays: Michael Snyder

This document discusses protein microarrays, which are high-density arrays containing hundreds to thousands of proteins. There are two main types: antibody microarrays, which use antibodies to profile protein expression, and functional protein microarrays, which screen for protein biochemical activities, interactions, and modifications. The document outlines methods for constructing functional protein microarrays using expression libraries to print entire proteomes. It summarizes several screens performed on a yeast proteome microarray that identified new protein-protein and protein-lipid interactions, kinase substrates, DNA-binding proteins, and drug targets. The document calls for establishing community standards around data reporting and repositories to facilitate data sharing and reproducibility.

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Protein Microarrays

Michael Snyder NIH Proteomics Standards Meeting, January 3, 2005

What is a Protein Microarray?

Many Things are Called Protein Microarrays or Chips


Affinity Resins for Fractionating Proteins (Ciphergen) Antibody Arrays Sets of Individual Proteins Protein Lysate Fractions Lysates from Tissues

What is a Protein Microarray? A high density array containing 100s to many thousands of proteins positioned in an addressable format

Many Things are Called Protein Microarrays or Chips


Affinity Resins for Fractionating Proteins (Ciphergen) Antibody Arrays Sets of Individual Proteins Protein Lysate Fractions Lysates from Tissues

Two Major Types of Protein Microarrays


1) Antibody Microarrays - Protein Profiling

Antigens

Cytokine Detection withAntibody Microarray

Microarray assay of a human serum sample. A 15 L sample of human serum was incubated for 30 min on a microarray with 75 different anticytokine antibodies printed in quadruplicate. Following washing and incubation with a mixture of secondary antibodies to each cytokine, detection was carried out using RCA. The fluorescent image was obtained using GenePix software on an Axon Microarray Scanner. The enlarged image shown represents one-eighth of the data acquired from a 1' 3' microscope slide. Fluorescent intensities are represented in pseudocolor, with lowest intensities in blue and highest intensities in white.

Many Commercial Antibody Arrays Are Available Arrays usually have 6-75 Antibodies Often Detect Cytokines Examples: - BD Biosciences - Scheichler & Schuell - Zyomyx

Profiling Types of Arrays


Antibodies - Rabbits, Mice Phage Display-Present Peptides on Bacteriophage Nucleic Acid Aptamers In vitro Selection

Major Challenge With Antibody Arrays 1) Antibody Specificity Haab et al. 20% of Antibodies Were Specific 2) Quantification

Two Major Types of Protein Microarrays


2. Functional Protein Microarrays

Protein Biochemical Activities Protein Modification and Regulation

Protein-Protein Small Molecule Enzymatic Interactions Interactions Assays


ATP ADP

Protein Pathways Drug Discovery and Development

Whats Needed to Make Functional Protein Arrays


Expression Library Methods for Purifying Many Proteins Array Technology

Cloning & Expression Strategy


Yeast ORFs
Primer 1 ATG TAA Primer 2

GAL

GST ORF

Gst::Orf

There are Many Expression Systems


E. coli Yeast Bacullovirus Plants Cell Free Systems (In vitro transcription/translation)

Printing the Yeast Proteome


GST:P1 GST:P2 GST:P3

Source Plate

Protein-Protein

Protein-Lipid

Protein-DNA

Glass Slides
1) Random Attachment
O O C-H C-H NH2 H C N
Schiff Base

Aldehyde Surface

2) Ligand Attachment

Screens Thus Far


20 Protein-Protein Interactions 8 Protein-Lipid Interactions 3 Nucleic Acids (dsDNA, ssDNA, polyA-mRNA) 4 Small Molecule Screens 3 Posttranslational Modifications 14 Antibodies 89 Kinase Probings

Biochemical Assays on Proteome Chips

Probe

-GST

Calmodulin

PI(3)P

PI(4,5)P2

Functional Protein Arrays Commercially Available


1) Yeast proteome 2) Human 2K array

General Issues for Standardization


1) Protein content & array platforms vary widely (Expression systems; slides, etc) 2) Protein quality may vary from prep to prep. 3) Negative results harder to interpret than DNA arrays

General Issues for Standardization (cont.)


4) Ideally, measurements should be quantitative Ab arrays - Each Ab must be standardized Func. Protein Array - like to get affinity constants 5) Field is still maturing

Informal QC Standards That Currently Exist:


Antibody Arrays: Immunoblot Analysis Functional Protein Arrays: Assess Protein Levels and Purity Validation Provide Hit List

Formal Community Standards That Currently Exist:

PEDRo Schema

Formal Community Standards That Should Exist:


People should have access to all primary and minimally processed data. Source, quality and amount of the proteins (e.g. Antibodies) should be documented. Results need to be validated statistically or by other means.

Recommendations
1) Need to Establish a Data Repository (analogous to GEO) 2) Need to Establish Mimimum Reporting Standards (MIAME) 3) Interactions should be deposited in a public database (e.g. BIND)

Comparison Between Protein and DNA Arrays


Funct. Protein array Common problems Aim Design Probes Array features Color Factors for the intensities Non-specific binding Varied; e.g.Binding assay Diverse formats: Surfaces; prot. source Pure Uneven amount Mostly one channel 1. 2. Binding affinity Feature amount on the array DNA array Specific probe amount Few standard formats Mixture Even amount One or two channels Amount of specific probes Trivial Smudges, uneven probing (position artifacts)

-Sticky tags -Anti-spots

Non-specific binding
All spots react

Anti-spots

- Raw intensities can be misleading - Negative control is necessary - Subtract signals from neighboring spots; normalize to negative control probings - Negative signals - Blocking problems - Reprobing

Next Steps
Have protein microarray experts establish standards in conjunction with the microarray community Deposit interactions in database Start discussions now

Kinase Assays on Protein Chips

Anti-GST

AutoPhosphorylation

Tpk1

Antibody Probing of the Yeast Proteome Microarray


Antibody # of +s 1 Monoclonal (3 Yeast + 3 Control) -Sed3, -Cox4 4 -Pep12 Anti-Peptide Polyclonal (6) Anti-FL Protein Polyclonal (2) -Hda1 -Mad2 -Nap1 -Cdc11 8 1 1770 7

Nap1

Cdc11 Sed3

Anti-Nap1

Mad2

-Sed3p Protometrix

Mad2

Software Issues
DNA array software can be used but the protein chips often have unique features not present in DNA arrays

Phosphorylome Network

Yeast Phosphorylome Map


122 Protein Kinase Homologs

- 14 Uncharacterized - 50% Have no known in vivo substrates


- <160 Known kinase-substrate phosphorylations

In Vitro Phosphorylome Summary


ARK1 (8) CKA1(26) CBK1 (1) ELM1 (5) ERK2 (43) GIN4 (5) HRR25 (13) KIN1 (3) KIN2 (28) KNS1 (8) MEK1 (33) PBS2 (6) PKK2 (5) PHO85-ALONE (6) PHO85-PCL1 (4) PHO85-PCL2 (9) PHO85-PCL9 (11) RIM11 (19) SLT2 (8) STE11 (2) STE20 (100) SWE1 (7) TPK1 (130) TPK2 (30) TPK3 (82) VHS1 (16) YGL059C (101) YMR291W (1) YOL128C (9) FUS3 (8) PTK2 (202) IPL1 (2) YCK3 (1) YAK1 (4) PRR1 (7) PKA (41) PRR2 (24) SKM1 (26) SKS1 (27) CDC5 (21) CLA4 (30) CTK1 (10) MKK1 (12) CDC15 (18) CDC28-Clb5 (56) DBF2 (85) IME2 (74) PAK1 (17) RAD53 (32) YGR052W (10) KSP1 (190) YCK1 (85) IRE1 (88) HAL5 (35) SAT4 (23) SSK22 (25) MCK110 (112) DUN1 (31) YKL171W (53) IKS1 (19) FUN31 (27) KIN28 (14) PRK1 (61) RCK2 (46) CMK2 (14) KCC4 (10) KIN4 (30) YOR267C (20) BCK1 (85) SRB10 (15) HSL1 (59) YPL141C (66) YDR466W(11) RIM15 (26) RIM15dead DBF2dead HSL1dead RAD53dead

Conclusions
1) Construct protein microarray containing nearly an entire proteome 2) Screen for diverse activities: interactions with proteins, DNA, small molecule; antibody specificity; kinase substrates 3) Unbiased screens yield unexpected results. Examples:
- Arg5,6 - Many novel substrates of kinases

4) Construct an in vitro phosphorylome map

Advantages of Protein Chips


Can screen many proteins simultaneously Small amounts of proteins and reagents High throughput Diverse applications-biochemical assays, posttranslational modifications, small molecule screening

Disadvantage
In Vitro Assay

Calmodulin-Binding Proteins
12 Known or Suspected Targets 33 New Binding Proteins Derived New Consensus Binding Site
14

R
P

L V
S T Q C

K H

G N

K
R
R

K S
G F E

K I L S I Q
V V

R
L S F Y I S

V N A K

L
E

D N

YFL003C/M S H4 YJR073C/O PI3 YB R050C/RE G2 YNL202 W/SPS19 Y OL016C/C M K2 YB R011C/ PP1 I

LK E T L Q S VK SL K D A L HS V D L Q S SK FQ L A I V DE H F I Q R LP ST R L N S AK I P L Q R LG ST R D I A DD L R L Q S QK KG G E L T LN P I I Q D TK KG K L R F

Summary of Genomic DNA Screen ~200 Proteins bound DNA probe 8 Novel ChIP chiped
5 No loci enriched 3 Showed enrichment: Mtw1, Dig2, Arg5,6

Identification of Drug Targets


Nutrient Rapa Drug (SMIR)
Tor1/2p

Fpr1p

???

Translation Glycogen Accumulation Arrest

G1 Arrest

J. Huang, H. Zhu, S. Schreiber, M. Snyder

SMIR3 8 Targets SMIR4 30 Targets

Human Allergen Microarray

Global Analysis of Kinase Substrates


Overexpress and Purify Kinase
30oC
ATP ADP

Incubate with Kinase and 33P-ATP Proteome Array Expose to Film

Wash

In Vitro Phosphorylome Summary


84 unique kinases and several isoforms with different
cyclins for 89 specific hit lists 3291 total phosphorylation events on 1238 individual targets On average kinase phosphorylated 48 proteins on chip (Range 1- 202) Most substrates were phosphorylated by only one kinase Identified at least 13 known kinase-substrate phosphorylations

Identification of New DNA Binding Activities


Cy3 labeled genomic DNA Probe proteome chip

~200 Positives

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