Subject:notes

Get your own FREE website, FREE domain & FREE mobile app with Company email.  Know More >
Protein Arrays
 

- Jobs Protein Arrays Resource Page

review | literature | meetings | companies
- EC Projects
- Databases Introduction
- Journals Array Basics Defining characteristics
- Mutagenesis &
Gene Disruption   Types of protein arrays
- Arrays & Chips Areas of application

- Proteomics Protein sources

- Structural Formats and surfaces

Genomics
Prtotein immobilisation
- Bioinformatic considerations
s Fabrication

  Detection

Capture arrays Binding molecules

  Detection systems
 
  Specificity and cross-reactivity

Related formats

  Applications of capture arrays

   

Protein arrays Protein in-situ arrays

 Protein functions and interactions

Library screening

Challenges/Bottlenecks Protein production and function

  Capture reagents and cross-reactivity

Array technology

Literature Guide Original papers

Reviews

Meetings

Companies

Introduction

Protein arrays are rapidly becoming established as a powerful means t

monitor their expression levels, and investigate protein interactions an
are seeing an explosive progress and interest at the moment and have
the most active areas emerging in biotechnology today. The objective
array development is to achieve efficient and sensitive high throughpu
carrying out large numbers of determinations in parallel by automated
were conceived originally as miniaturised immunoassays, further deve
driven by genome projects on the one hand and improved expertise in
recombinant proteins on the other. Protein arrays make possible the p
screening of thousands of interactions, encompassing protein-antibody
protein-ligand or protein-drug, enzyme-substrate screening and multia
assays. In the microarray or chip format, such determinations can be c
minimum use of materials while generating large amounts of data. Mo
proteins are made by recombinant methods, there is direct connectivit
on protein arrays and DNA sequence information.

At the present time, protein arrays are poised to become a central pro
technology, important both in basic research and commercially for biot
enterprises. It is well recognised that the complexity of the human pro
that of the genome. When variables such as alternative gene splicing e
translational modifications and individual coding variants are taken int
number of different molecular protein species in man is likely to be at
magnitude greater than the number of genes (about 21,000), i.e. poss
1,000,000 proteins. Proteomics investigations are at the leading edge
genomics today and the development of protein arrays reflects the rea
functional genomics discoveries will depend heavily on progress in defi
expression of, and interactions among, proteins. Conventional proteom
gel electrophoresis and mass spectrometry, while highly effective, has
particular may miss many proteins of interest when expressed at low a
unsuited to diagnostic applications . Since the low abundance proteins
the greatest diagnostic interest (e.g. cytokines and biomarkers in plas
therefore an acknowledged need for other highly sensitive, specific and
throughput technologies for protein detection, quantitation and differe
analysis in health and disease. For this reason, protein arrays are gene
interest at the research and biotechnology levels.
Microarray ELISA-style assays will accelerate immunodiagnostics signif
aspect of the technology was discussed in the 1980s by Ekins, who int
concept of the ambient analyte assay and demonstrated that microspo
could be perfomed with high sensitivity and selectivity. In addition to d
applications, protein array technology promises to accelerate basic
protein interactions and will allow protein expression profiling, ranging
numbers of proteins up to global proteomic analysis, while in the pharm
protein arrays can be integrated into target identification and validatio
However, as with other high throughput functional genomics technolog
major technical demands which will need to be solved in order to achie
capability. This page will attempt to keep abreast of developments in p
technologies and to provide an up to date guide to the relevant
and companies in the field.

(Note, in the text below, mention is made of specific

arrays; links to their websites can be found on the companies
literature references are not annotated in the text, a comprehensive
provided.)

Array basics

Defining characteristics of protein arrays

Protein arrays are solid-phase ligand binding assay systems using imm
on surfaces which include glass, membranes, microtiter wells, mass sp
and beads or other particles. The assays are highly parallel (multiplexe
miniaturised (microarrays, protein chips). Their advantages include be
automatable, capable of high sensitivity, economical on reagents, and
abundance of data for a single experiment. Bioinformatics support is im
handling demands sophisticated software and data comparison analysi
some of the software can be adapted from that used for DNA arrays, a
hardware and detection systems.

Types of protein arrays

There are three general types of protein array:
(a) large-scale functional chips ( target protein arrays
numbers of purified proteins and used to assay a wide range of bioche
such as protein-protein, protein-DNA, protein-small molecule interactio
activity, and to detect antibodies and demonstrate their specificity.
(b) the analytical capture arrays carry affinity reagents, primarily antib
also be alternative protein scaffolds, peptides or nucleic acid aptamers
detect and quantitate analytes in complex mixtures such as plasma/se
extracts;
(c) lysate (reverse protein) arrays in which the complex samples such
are printed on the surface and targets then detected with antibodies o
 Areas of application
All three array types have uses in diagnostics (biomarkers or antibody
discovery research. T he capture arrays, are used to detect target mol
such as plasma or tissue extracts. Typically a complex mixture would b
of possibly thousands of specific binders, and the individual bound ana
parallel by appropriate labelling and scanning. In diagnostics, capture
to carry out multiple immunoassays in parallel, both testing for severa
individual sera for example and (by segmenting the array) testing man
simultaneously. In proteomics, capture arrays are used to quantitate a
levels of proteins in different samples in health and disease, i.e. protei
profiling.
Target protein arrays are used in for in vitro functional interaction scre
particularly to detect antibodies in individual patient or animal sera, du
monitor immune responses. The capture reagents themselves will nee
and screened for cross reactivity against many proteins, which can als
multiplex array format against multiple protein targets.
Lysate arrays allow thousands of samples to be analysed simultaneous
platform, greatly increasing throughput and simplifying quantitative an
samples. Furthermore, an exceedingly small amount of sample is requ
the arrays, thus permitting the analysis of rare and valuable patient sa

Thus, broadly speaking, there are at least four major areas where prot
being applied, each of which requires appropriate formats and readout
1. Diagnostics: detection of antigens and antibodies in blood samples
to discover new disease markers; environment and food monitoring. A
autoimmunity, allergy and cancer are listed in the tables below.
2. Proteomics: Protein expression profiling
3. Protein functional analysis: protein-protein interactions; ligand-b
of receptors; enzyme activities;
4. Antibody characterisation: cross reactivity and specificity, epitop
 

Protein sources
For construction of arrays, sources of proteins include cell-based expre
recombinant proteins, purification from natural sources, production
translation systems, and synthetic methods for peptides. Many of thes
automated for high throughput production. For capture arrays and pro
analysis, it is clearly important that proteins should be correctly folded
this is not always the case, e.g. where recombinant proteins are extrac
under denaturing conditions or where the surface encourages unfoldin
hand, arrays of denatured proteins are useful in screening antibodies f
identifying autoantibodies and selecting ligand binding proteins , where
are recognised .

Formats and surfaces

Protein arrays have been designed as a miniaturisation of familiar imm
such as ELISA and dot blotting, often utilising fluorescent readout, and
robotics and high throughput detection systems to enable multiple ass
out in parallel. Commonly used physical supports include glass slides,
nitrocellulose or PVDF membranes, and magnetic and other microbead
microdrops of protein delivered onto planar surfaces are the most fam
are a number of more advanced architectures incorporating developme
microfluidics and nanotechnology. Particles in suspension can also be u
arrays, providing they are coded for identification [e.g. Luminex, Bio-R

Protein immobilisation considerations

Variables in immobilisation of proteins include both the coupling reage
of the surface being coupled to. The properties of a good protein array
are that it should be chemically stable before and after the coupling pr
good spot morphology; display minimal nonspecific binding; not contri
in detection systems; and be compatible with different detection syste
immobilisation method used should be reproducible; applicable to prot
properties (size, hydrophilic, hydrophobic); amenable to high throughp
automation; and compatible with retention of fully functional protein a
of the surface-bound protein is recognised as a possible factor in prese
or substrate in an active state; for capture arrays the most efficient bi
obtained with orientated capture reagents, which generally require site
of the protein.

Both covalent and noncovalent methods of protein immobilisation are

various pros and cons. Diffusion into porous surfaces is a successful m
noncovalent binding of unmodified protein within hydrogel structures,
dimensional polyacrylamide gel; these substrates are reported to give
background on glass microarrays, with a high capacity and retention o
Passive adsorption to surfaces is methodologically simple, but allows li
orientational control; it may alter the functional properties of the prote
unfolding, and reproducibility and efficiency are variable. Covalent cou
provide a stable linkage, can be applied to a range of proteins and hav
reproducibility; however, orientation may be variable, chemical deriva
the function of the protein and requires a stable interactive surface. Bi
methods utilising a tag (such as hexahistidine/Ni-NTA or biotin/avidin)
provide a stable linkage and bind the protein specifically and in reprod
but the partner reagent must first be immobilised adequately on the su

Fabrication
Array fabrication methods include robotic contact printing, ink-jetting,
spotting and photolithography. A number of commercial arrayers (spot
as well as manual equipment. The novel methods for production of pro
(see section below) avoid the need to express and purify the proteins,
the DNA templates from which proteins are expressed on the array its
transcription/translation systems.

At the limit of spot size and density are nanoarrays, with spots on the
spatial scale, enabling thousands of reactions to be performed on a sin
1mm square.

Detection
Fluorescence labelling and detection methods are widely used and are
The same instrumentation as used for scanning DNA microarrays is ap
arrays. For differential display, capture (e.g. antibody) arrays can be p
fluorescently labelled proteins from two different cell states, in which c
directly conjugated with different fluorophores (e.g. Cy-3, Cy-5) and m
the colour acts as a readout for changes in target abundance. Use of 2
direct labelling allows control for spot variability as well as comparison
different samples. Fluorescent readout sensitivity can be amplified 10-
tyramide signal amplification (TSA). Planar waveguide technology enab
fluorescence detection, with the additional advantage of no intervening
procedures. High sensitivity can also be achieved with suspension bead
using phycoerythrin as label or the properties of semiconductor nanocr
dots). A number of novel alternative readouts have been developed, in
adaptations of surface plasmon resonance (for real time kinetic measu
circle DNA amplification (for sensitivity down to near the single molecu
spectrometry (for definitive protein identification), resonance light scat
force microscopy (for nanoarrays).

 Capture arrays

These form the basis of diagnostic chips for detection of clinically impo
as biomarkers, and arrays for expression profiling. They employ high a
reagents, including conventional and recombinant (single chain) antibo
domains, engineered scaffolds, peptides or nucleic acid aptamers, to b
specific target ligands in high throughput, parallel manner.

Binding molecules
Antibody arrays have the required properties of specificity and accepta
and a number are available commercially for different sets of antigens
signalling pathways. Antibodies for capture arrays are made either by
immunisation (polyclonal sera and hybridomas), or as recombinant sin
fragments (scFv), usually expressed in E. coli, after selection from pha
display libraries. In addition, single V-domains from camelids or engine
equivalents may also be useful in arrays.

The term 'scaffold' refers to protein domains which are engineered into
capable of binding diverse target molecules with antibody-like properti
and affinity. The variants can be produced in a genetic library format a
against individual targets by phage, bacterial or ribosome display. Suc
or frameworks include Designed Ankyrin Repeat proteins (DARPins), A
based on Staph. aureus protein A, Trinectins based on fibronectins and
on the lipocalin structure. These can be used on capture arrays in a sim
antibodies and often have advantages of robustness and ease of produ

Nonprotein capture molecules, notably the single-stranded nucleic acid

bind protein ligands with high specificity and affinity, are also used in a
are selected from libraries of oligonucleotides by the Selex� procedure
interaction with protein can be enhanced by covalent
of brominated deoxyuridine and UV-activated crosslinking (photoaptam
Photocrosslinking to ligand reduces the crossreactivity of aptamers due
steric requirements. More recently, SomaLogic introduced �Slow� Ap
�SLaptamers�, selected for a slow rate of dissociation (t1/2 >30mins
hundred specificities hacve now been selected. Aptamers in general ha
of ease of production by automated oligonucleotide synthesis and the
robustness of DNA.

Detection systems for capture arrays

 What is required from any method is optimal sensitivity and specificity
background to give high signal to noise. Since analyte concentrations c
sensitivity has to be tailored appropriately; serial dilution of the sampl
antibodies of different affinities are solutions to this problem. Sensitivi
important where the proteins of interest are in very low concentration
extracts, requiring detection in the pg range or lower, such as cytokine
expression products in cells.
Protein analytes binding to antibody arrays may be detected directly o
antibody in a sandwich assay. Direct labelling is used for comparison o
with different fluorophores. Where pairs of antibodies directed at the s
are available, sandwich immunoassays provide high specificity and sen
therefore the method of choice for low abundance proteins such as cyt
give the possibility of detection of protein modifications. Label-free det
including MS and SPR, avoid alteration of ligand.

Specificity and cross-reactivity on capture arrays

The question of cross-reactivity is an important one which applies to a
and particularly to antibodies, being the most popular reagents. While
thought of as being highly specific, monoclonals can show unpredictab
which will be revealed by thorough screening. The ultimate usefulness
reagents then depends on the relative level of cross-reaction and spec
important general principle is that, for optimal specificity where assays
multiplexed, it is essential to provide dual level target recognition, i.e.
specificity for each locus in the array. Sandwich assays achieve this wi
photocrosslinking reduces the cross-reactivity of aptamers and MS pro
label-free protein identification.

The use of sandwich assays (above), in which antibody pairs are used
ligand, go a long way towards eliminating the problem, since it is unlik
members of the sandwich will exhibit the same cross-reactivity. Never
multiplexing of sandwich assays is limited to around 40 reactions, aga
reactivity of the reagents. A related technology in which pairs of binde
same target protein is the proximity ligation method which, combined
amplification, can be adapted for highly sensitive and specific protein a
format.

Related formats
Another methodology which can be used diagnostically and in expressi
ProteinChip� array [Ciphergen], in which solid phase chromatographic
proteins with similar characteristics of charge or hydrophobicity from m
plasma or tumour extracts, and SELDI-TOF mass spectrometry is used
retained proteins. The ProteinChip� is credited with the ability to ident
markers. However, this technology differs from the protein arrays und
since, in general, it does not involve immobilisation of individual protei
specific ligand interactions.
Applications of capture arrays

1. Diagnostics
As diagnostic devices, microarrays exploit the power of multiplexing si
analyses of different samples and repeated analyses of the same samp
formats include arrays of antibodies, as in detection of cytokines [e.g.
Zeptosens, Molecular Staging, Luminex], and antigens to detect serum
screens for infections, autoimmune diseases and allergies. Highly para
arrays will allow determination of tumour markers in extracts with only
biopsy material, creating new possibilities for monitoring cancer treatm
Discovery of new autoantibody specificities is possible by screening pa
arrays of human proteins [Protagen].
While diagnostic arrays have tended to be of relatively low density and
specific assay purposes, they have high throughput potential through a
analysis and microfluidics.

2. Proteomics: Protein expression profiling

The quantitative detection of proteins in cells and tissues and comparis
conditions (health, disease, differentiation, drug treatment, etc) is a ce
proteomics. The array format is well established for the rapid, global a
acids, as in the use of oligonucleotide and cDNA arrays for gene expre
(transcription) profiling. However, mRNA expression data has acknowl
shortcomings as an indicator of actual protein abundance or dynamics,
reveals nothing about post-translational modifications or protein-prote
Two-dimensional gel electrophoresis technology, on which most proteo
based currently, is also limited in various ways, particularly in the diffi
quantitatively estimating low abundance proteins. For information abo
of the proteome, protein and peptide arrays are becoming major tools
information that will be obtained from them in the future will complem
data. Capture arrays sensitively and accurately detect low levels of pro
technical know-how on the part of the user and we can expect them to
measure differential protein expression. They will provide a powerful a
platform for extending molecular analysis beyond the limitations of DN
assumes that the necessary numbers of antibodies or other capture re
specificity and affinity can be obtained against the proteins of interest
against the entire proteome. There are ambitious binder project projec
stage to fill this need (e.g. ProteomeBinders).

  

A format for differential protein expression profiling using antibody arr

figure above. A mixture (e.g. of two tissue extracts) is applied to the a
analytes of interest are captured by the specific ligand binders, followe
binding. Similar to comparison of samples from normal and diseased t
arrays or on 2D gels, reference and test samples can be labelled with C
mixed, gel filtered to remove unbound dyes and then incubated on a c
antibodies. Increased or decreased protein expression is assessed usin
up- or down-regulated proteins can be identified from the ratios of the
familiar 'traffic light' (red, yellow, green) system. Here, directly labelle
derivatised) samples are used, but there are a number of alternative d
strategies.

Protein arrays

Functional chips have been constructed by immobilising large numbers

proteins and used to assay a wide range of biochemical functions, such
interactions with other proteins, drug-target interactions, enzyme-subs
Generally they require an expression library, cloned into
which the expressed proteins are then purified, e.g. via a His tag, and
free protein transcription/translation is a valuable alternative for synth
which do not express well in bacterial or other in vivo systems.

In situ protein arrays

Due to the sensitivity and hetergeneity of proteins, it is difficult to stor

protein arrays in a functional state for long periods of time. In contrast
stable molecule capable of long-term storage. Therefore, an interestin
make protein arrays directly from DNA, either co-distributed or pre-arr
free protein experssion systems, to create the proteins on the arrays o
when required. Moreover, since the proteins are made and immobilised
a single step on the chip surface, the laborious and often costly proces
protein purification and DNA cloning are avoided.

Thus, in situ (�on-chip�) methods address three issues in protein arr

efficient global protein expression and availability; (ii) functional prote
and purification; and (iii) on-chip stability over time. Three recently de
are termed PISA (protein in situ array), NAPPA (nucleic acid programm
and DAPA (DNA array to protein array).

PISA method
In PISA (He, M., Taussig M.J. Nucleic Acids Res 29, e73, 2001), protein
directly from DNA, either in solution or immobilised, and become attac
surface as they are made, through recognition of a tag sequence. The
expressed in parallel in vitro utilising a cell free system, commonly rab
E. coli S30, to perform coupled transcription and translation. The key f
method is that protein expression is performed on a surface which is p
immobilising agent capable of binding the tag. Thus after each protein
becomes fixed simultaneously and specifically to the surface and the o
be washed away. Starting from PCR DNA, the PISA procedure takes ab
create the protein array. Microarrays are produced directly onto glass
mixing the DNA with the cell free lysate system before spotting or by a
technique (MIST) in which DNA is spotted first followed by the express
(Angenendt P. et al, 2006).

NAPPA method
Transcription and translation from an immobilised (as opposed to a so
template is a further desirable development of on-chip technologies wh
conversion of DNA arrays to protein arrays. This has been initiated wit
Nucleic Acid Programmable Protein Array or NAPPA (Ramachandran N.
86-90, 2004). Biotinylated cDNA plasmids encoding the proteins as GS
printed onto an avidin coated slide, together with an anti-GST antibody
capture entity. The cDNA array is then covered with rabbit reticulocyte
the proteins, which become trapped by the antibody adjacent to each
proteins thereby becoming immobilised with the same layout as the cD
procedure was shown to work quite precisely, with discrete protein spo
diffusion or cross-talk, and used for functional studies of interactions b
proteins. Recently it was expanded to high density arrays of 1000 diffe
(Ramachandran et al. Nature Methods 5:535-538, 2008). Note that th
generates a protein array in which the immobilised proteins are presen
DNA and capture agent, and furthermore that the DNA array can only

  

DAPA method
This method for in situ protein arraying represents a further advance i
immobilised DNA array as the template to generate �pure� protein ar
surface from the DNA, and also is able to produce multiple copies of a
the same DNA template (He M, et al. Nature Methods, 5, 175-7, 2008)
synthesis is performed in a membrane held between two surfaces (gla
which is arrayed with DNA molecules while the other surface carries a
capture the translated proteins. Individual, tagged proteins are synthe
from the arrayed DNA, diffuse across the gap and subsequently immob
interaction with the tag-capturing reagent on the opposite surface to f
array. Discrete spots which accurately reflect the DNA in position and q
produced. Moreover replicate copies of the protein array can be obtain
DNA, and at least 20 repeats have been demonstrated.

 Screening for protein functions and interactions

For detecting protein-protein interactions, protein arrays can be in vitr
the cell-based yeast two-hybrid system and may be useful where the l
such as interactions involving secreted proteins or proteins with disulp
throughput analysis of biochemical activities on arrays has been descri
protein kinases and for various functions (protein-protein and protein-
of the yeast proteome, where a large proportion of all yeast open-read
expressed and immobilised on a microarray. Large-scale 'proteome ch
very useful in identification of functional interactions, drug screening, e
possible screen will be for the effect of polymorphisms arising from dis
coding SNPs (SAPs, single amino acid polymorphisms); such informatio
in ascertaining the effects of SNPs on drug responses and side effects
(pharmacogenomics). One restriction is that proteins which are only fu
multicomponent complexes will be more difficult to analyse on protein

Library screening
As a two-dimensional display of individual elements, a protein array ca
screen phage or ribosome display libraries, in order to select specific b
including antibodies, synthetic scaffolds, peptides and aptamers. In th
against library' screening can be carried out. Screening of drug candid
combinatorial chemical libraries against an array of protein targets ide
genome projects is another application of the approach.

Remaining Challenges and bottlenecks

Despite the recent progress, there are a number of important technica

bottlenecks in protein array technologies, some of which are unique to
others are common to high throughput methods in general, which will
in order to achieve the maximum capability. They include the problem
global, functional protein expression for array construction and selectio
binders, aspects of protein coupling to surfaces, the sensitivity and dyn
detection systems, and standardisation and data storage. At each bott
choices of alternative methods. Issues which need to be addressed inc

Protein production and function

A bottleneck in creating protein arrays, especially those which aim to b
production (expression and purification) of the huge diversity of protei
the array elements, including capture molecules. Collections of high-qu
clones for different species are required and systems for the productio
high throughput must be developed. The challenge will be to make exp
sufficiently comprehensive such that potentially all proteins (including
proteins) become available; different systems (bacterial, yeast, baculo
will be employed for different proteins. One aim will be to express man
for functional analysis, and another is to raise antibodies and other cap
against them for array production.

A further consideration for many purposes is that arrayed proteins sho

folded and functional. This will require extensive and almost individual
with proteins of unknown function may be hard to achieve! Production
immobilisation of membrane proteins, which comprise a large proporti
proteome, is a continuing difficulty; these may be best accessed in the
Proteome chemistry is also hugely complicated by the existence of freq
post-translational modifications (PTMs). The problem will be how to inc
which phosphorylation and glycosylation are just two of many, into pro
well as wanting to put modified proteins onto the chip surface, we wou
know the PTMs of captured proteins, which can be determined through
antibodies or mass spectrometry.

Capture reagents and cross-reactivity

A current limitation on capture arrays is the availability of the antibodi
proteins, etc.), especially where pairs are required against each target
assays. Accessing very large numbers of affinity reagents is a major ch
Automated screening of antibody or scaffold libraries against arrays of
may be the most rapid way of developing the thousands of reagents re
 proteome expression profiling. There is some discussion over whether
antisera, hybridomas or selection from recombinant library systems is
forward, but in practice, providing they are screened thoroughly for cr
(below), products of all three are used successfully in the array format

The design of capture arrays, particularly when exposed to heterologo

as plasma and tissue extracts, needs to take into consideration the pro
reactivity which will occur particularly with highly multiplexed assays.
surprisingly cross-reactive, which in the high throughput microarray fie
results misleading or, at worst, useless. Successful multianalyte analys
require careful screening of each polyclonal antiserum, hybridoma or r
for cross-reactions against all antigens on the array. The use of combin
antibodies against individual targets in sandwich assays should help to
reactions. The importance of dual level target recognition, ensuring tw
specificity for each element in the array, has been noted. This can be a
combinations of antibodies against individual targets in sandwich assay
ligation, or through linkage to mass spectrometry to confirm the identi
ligands.

Array technology
What are the best coupling chemistries and supports? There are sever
in both categories (see Table above). Comparisons of different system
becoming available. The stability and lifetime of protein arrays in differ
to be considered; protein arrays are likely to be far less robust than DN

Detection methods are another important consideration, with requirem

accuracy and quantitation over a wide range. The design of the array w
by the readout system.

Finally, standardisation is an issue common to all high throughput tech

existence and development of many alternative formats and conditions
to problems in comparison of results. Standards for protein arrays and
their implementation will need to be established at an international lev

Comments on this webpage and suggestions for improvement are welcome. Please e-mail the
Page last updated 20 May 2009.

� Mike Taussig & Oda Stoevesandt, 2009

 
 
                         

   

Copyright © 2015 Rediff.com India Limited. All rights reserved.

Disclaimer | Privacy Policy | Help | Write to us