Novel technologies and the remaining challenges

Microarray- and macroarray-based protein assays

Nucleic acid amplification using PCR has revolutionized forensic science by providing a powerful tool for mitochondrial and chromosomal DNA typing and other nucleic acid-based analyses. Hundreds of publications available so far report the use of PCR amplification in forensics and related disciplines. DNA

microarrays are another relatively new technology that allows a much higher multiplexity of nucleic acid analysis (by hybridizing a probe simultaneously to many thousands of spots of various cDNAs or oligonucleotides in a single array). Today DNA microarrays have become a routine tool in transcriptomics, but have not so far been used widely in forensic science, where PCR remains the preferred tool (due to its sensitivity). One of the difficulties is that DNA analysis using microarrays requires orders of magnitude larger amounts of initial material to be used compared to PCR-based analysis, therefore if only little material is available, which is often the case in forensic applications, microarrays may be unsuitable for the job. For example, PCR sensitivity is ultimately one DNA molecule, whilst microarrays (i.e. hybridization analysis) would typically require several hundred nanograms of mRNA. Protein microarrays would seem to be an obvious successor to DNA arrays and many formats have already been attempted. Unlike nucleic acids, however, protein targets are typically non-homogeneous and affinity capture agents are often poorly characterized, making the experiments difficult to perfect and reproduce. Moreover, running multiple affinity assays in parallel (multiplexing) is not possible due to the heterogeneity of antibody affinities to their protein targets.

Unlike genomic DNA, which is present at one of two copies per cell, proteins are often present in millions of copies per single cell. This could make a protein affinity assay (which is analogous to DNA hybridization) possible in cases where DNA microarrays may fail. There is very little, if any, research published so far on the use of protein microarrays in forensics, and this is not surprising taking into account the technical difficulties of working with immobilizing proteins in their affinity-active conformational states.

Protein array-based proteomics has many advantages over traditional pro-teomic techniques based on two-dimensional gels and chromatography (Soloviev and Terrett, 2005). Highly multiplexed assays can be achieved by spatially separating antibodies on the membrane when spotting, and standard curves can be plotted for a range of known sample antigen concentrations, from which unknown sample concentrations can be derived. An experiment with a single protein chip can supply information on thousands of proteins simultaneously and this provides a considerable increase in throughput. A micro- or macroarray is compatible with both competitive and non-competitive immunoassay formats:

1. Competitive:

a. Fluorescently labelled reference competes with sample antigen for antibodies discretely spotted on solid support. Fluorescence intensity is inversely proportional to sample antigen concentration.

b. Fluorescently labelled antibody is added to sample and then hybridized with reference antigen discretely spotted on solid support. Fluorescence intensity is directly proportional to sample antigen concentration.

2. Non-competitive:

a. Fluorescently labelled sample binds to antibody discretely spotted on solid support. Fluorescence intensity is directly proportional to sample antigen concentration.

b. Fluorescently labelled antibody (primary or secondary) binds to sample antigen discretely spotted on solid support. Fluorescence intensity is proportional to sample antigen concentration.

A novel modification of the competitive displacement strategy has been reported recently (Barry et al., 2003) and is a technique that can utilize almost any antibody or indeed other types of affinity reagents or their mixtures (affinity heterogeneity is not an issue) and allows a high degree of multiplexing (the number of proteins assayed being limited only by antibody availability). It is tolerant towards high levels of non-specific binding and does not require any potentially interaction-disrupting labelling of the experimental samples. It is capable of quantitative comparison of unlabelled experimental samples over a wide concentration range. Other advantages of the method include its relative simplicity and low cost (only a single labelled reference sample per series and a single array per sample are required), and intrinsic signal normalization and compatibility with known signal amplification techniques (e.g. ELISA, electrochemical luminescence, RCAT, DNA fusions, etc.); see Soloviev et al., 2004, and Barry and Soloviev, 2004, for a more detailed review and additional references).

Affinity and combinatorial peptidomics approaches

Affinity peptidomics is the most generic affinity assay system reported to date, which is applicable for protein identification and quantification for forensic and biometric applications. It is multiplatform compatible (liquid chromatography, microarrays, microfluidics and mass spectrometry). In the peptidomic approach the assayed mixture of proteins (typically a mixture of heterogeneous proteins) is enzymatically digested (e.g. with trypsin) prior to affinity capture to form a homogeneous mixture of short peptides. These peptides can also be predicted by in silico digestion of individual proteins or protein databases. Capture agents can therefore be specifically designed for all or a subset of suitable peptides from each of the proteins. Such peptides are fully predictable on the basis of protein sequence alone (or even predicted sequence). The use of mass spectrometry (e.g. MALDI-ToF-MS) for a direct confirmation of the identity of the species captured provides an additional advantage compared to the more usual method of detection in which fluorescently labelled captured species are scanned to give a spatially resolved image of the array.

In peptidomics, each protein is broken down into many smaller components, resulting in the availability of a large range of peptides with less heterogenic physical and chemical properties (which are also more predictable). Large numbers of proteolytic peptides allow multiple independent assays for the same protein target to be performed, thus also increasing the reliability of the assay. Peptidomics enables a high-throughput screening of proteins (e.g. in a microarray format) and has several advantages over the affinity capture of intact proteins:

1. As peptides are much more stable and robust than proteins, protein denatura-tion and degradation is not an issue since only one or a few intact peptides would be required for the analysis. Affinity peptidomics does not have to struggle with unstable or degraded proteins, it uses proteolytically digested samples and relies on anti-peptide affinity reagents (e.g. antibodies, but these can also be antibody mimics (Nord et al., 1997), molecularly imprinted reagents (Haupt and Mosbach, 1998), etc.

2. Peptides are also particularly suited for detection by mass spectrometric techniques, such as MALDI-ToF-MS, for direct analysis of samples on a solid substrate such as microarrays. The peptide mass range is such that isotopic resolution is easily achieved and hence fully quantitative analysis is possible (e.g. using isotopically labelled standards as in AQUA (Gerber et al., 2003), MCAT (Cagney and Emili, 2002) or ICAT (Gygi et al., 1999) approaches.

3. Digestion of cellular fractions or even intact tissues results in the release of peptides, which in most cases will contain more than one hydrophilic peptide (Soloviev and Finch, 2005) per protein, thus improving the assay.

4. Antibody can be against linear unfolded fragments, not native folded proteins, and therefore peptide 'antigens' can be more easily generated, such as by chemical synthesis of in silico predicted peptides (as opposed to traditionally used fully folded proteins or their fragments) (Soloviev et al., 2003).

5. Such affinity reagents can be obtained at lower costs and in a truly high-throughput manner and against most antigenic peptides, and their specificities and affinities can be more easily controlled.

6. The affinity peptidomics approach is suitable for both microarray-based assays (for quick/routine applications, whether field or laboratory-based) and analytical mass spectrometry-based analysis (e.g. quantitative mass spec-trometry using AQUA / ICAT / MCAT approaches), suitable for resolving difficult cases or independent confirmation of the array data if required.

Another approach suitable in principle for use in forensic applications is the combinatorial approach to peptidomics analysis (Soloviev and Finch, 2005). It utilizes the original peptidomics approach where protein samples are proteolyti-cally digested using one or a combination of proteases, but in place of affinity purification the peptide pool is depleted through selective chemical binding of a subset of peptides to a solid support. This combinatorial approach utilizes the selective chemical reactivities of the side chains of individual amino acids, and thus is the nearest to a sequence-dependent analysis (e.g. DNA-based analyses). Together, the affinity peptidomics (fast and high throughput) and the combinatorial approaches (more sequence-dependent analysis) provide a viable alternative to traditional protein analysis techniques (protein preservation-separation pathway). Peptidomics approaches provide the most generic protein assay system to date, applicable for protein identification, quantification and expression profiling. These are multiplatform compatible and are capable of the analysis of partially degraded proteins, which makes them especially suitable for forensic applications. Combining the peptidomics approach with a protein microarray platform will eventually yield a new miniature tool for on-site analysis and scene-of-crime applications.

Was this article helpful?

0 0

Post a comment