**5. Protein microarrays**

the inherently high gel-to-gel variability and relatively low resolving power of individual gels impacts on the accuracy of spot picking and imposes a limitation due to co-migrating proteins, which is especially problematic for low-abundance protein targets. Several modifications have been suggested to address such limitations, including multi-colour fluorescence-based 2-D gel immunoproteomics approaches [62], but these still do not address the fundamental issues of the limited resolving power of the gels, the modest limit of detection or the throughput for SERPA. This technology is thus less widely used for autoantigen discovery now and has been largely supplanted by newer technologies that are better able to overcome these limitations.

SEREX is one of the oldest methods for autoantigen discovery and utilises human cDNA expression libraries to profile autoantibody repertoires. The methodology for SEREX initially involves generation of a cDNA library from a cancer tissue or tumour cell line, followed by cloning of that library into a suitable expression vector, clonal separation of the library and expression of the encoded proteins in *Escherichia coli* cells grown on solid media. Colonies are then transferred to a nitrocellulose or PVDF membrane, lysed and the expressed recombinant proteins blotted with sera from patients and healthy controls. Sero-reactive proteins are then identified by sequencing the cDNA from positive colonies [63], which makes it more sensitive than the SERPA method since the latter relies on direct protein identification and is therefore limited by absolute protein abundance. Furthermore, the clonal separation of the members of the cDNA expression library provides greater resolving power than the gel-based SERPA method. However, as with all library-based screening methods, over-sampling is required to ensure that all members of the library are examined in SEREX: thus, if for example the cDNA

complete coverage, so even with the advent of colony picking and arraying robots, SEREX

The first cancer testis antigen, NY-ESO-1, was identified by SEREX by analysing tumour associated antigens (TAAs) that elicited a high titre IgG antibody in sera from patients with different types of cancer [64]. SEREX has also been successfully used to identify several TAAs that generate a humoral immune response in cancers such as those from the kidney, lung, breast and colon [63]. However, a fundamental limitation of SEREX is that the method lacks the ability to differentiate or detect post-translational modifications (PTMs) that are likely to play a significant role in autoimmune diseases [62] and cancers [63]. This approach also restricts the types of TAAs identified to those that can be expressed in a prokaryotic system and also effectively excludes TAAs that require folding mechanisms unique to eukaryotes to achieve the correct conformational epitope for recognition [63]. SEREX may also miss TAAs that are represented by truncated cDNAs in the library, since the encoded protein may lack specific epitopes or even whole domains. Furthermore, identification of TAAs is inherently limited to those that were expressed by the specific patient tumour or cell line from which the cDNA library was derived, which means that more than one cDNA library may be required to identify comprehensive set of TAAs for different cancers [63]. In addition, the presence of the crude prokaryotic cell lysate in every spot can give rise to high background binding in SEREX

colonies would need to be screened for

**4.5. Serological analysis of recombinant cDNA expression libraries (SEREX)**

unique clones, then at least 10<sup>5</sup>

library contains 10<sup>4</sup>

172 Autoantibodies and Cytokines

remains a relatively low throughput method.

Protein microarrays are a versatile, miniaturised platform used to simultaneously characterise the biomolecular interactions of thousands of different proteins that are spotted in defined locations on a solid support; as such, protein microarrays represent a natural technological evolution from ELISA, SERPA and SEREX. Protein microarrays in principle allow the quantitative analysis of binding of a wide variety of analytes - including antibodies, proteins, DNA, RNA, small molecules, lipids, enzymes as well as peptides - to the arrayed proteins. The three types of protein microarrays that are commonly used are analytical, functional and reverse-phase microarrays. Analytical protein arrays, or antibody arrays, are ideal for quantification of different known proteins in a biological sample, monitoring protein expression levels and protein profiling in what amounts to miniaturised, highly multiplexed ELISA assays. Functional protein microarrays can be sub-divided into those based on recombinant proteins and those based on native proteins and can be used for autoantibody and immune response profiling, biomolecular interaction profiling and identification of enzyme substrates, amongst others [1]. Reverse-phase protein arrays are comprised of spots of different crude tissue homogenates or cell lysates and are suited for detection of known proteins in multiple tissues/cells based on blotting of the reverse-phase arrays with antigen-specific antibodies. In general, protein microarrays can be applied in diagnostic and therapeutic research, through new biomarker discovery for disease staging and monitoring, potential drug-target evaluation and for identification of new drug targets. Of the different protein array types, functional protein arrays appear best suited to autoantigen discovery and autoantibody profiling and are discussed in more detail below.

#### **5.1. Recombinant protein production**

Different protein production systems can be employed to produce recombinant proteins in sufficient quantities for protein microarray fabrication. The key problem associated with recombinant protein production is identifying the best expression system for a particular protein. To date, there is no universally applicable protein expression system [65]. Each system has its advantages and disadvantages; therefore, the choice of expression system selection should be based on the properties of the recombinant protein as well as the scale of expression required. Although exploring multiple expression systems in parallel sounds enticing, factors such as protein solubility, yield, speed and cost need to be taken into consideration as it involves substantial resources. Choosing the right system for protein expression can be particularly important in obtaining biologically active and functional recombinant proteins [1].

Bacteria, notably *E. coli*, represent the most commonly used expression systems for protein production since they give high protein yields at a relatively low cost, require simple and rapid culture conditions, and are highly scalable. In addition, many parameters can be altered to optimise expression levels of protein. However, inefficient disulfide bond formation, insolubility, aggregation and poor folding of proteins have been reported using this method, as well as very minimal capability in performing post-translational modifications [65].

gives rise to the second issue - the loss of protein conformation upon immobilisation. In particular, when the functional domains interact excessively with a solid surface, the orientation of the proteins may be altered or completely lost, resulting in the subsequent disruption of the functional domains and loss of discontinuous epitopes [66]. Partial or complete denaturation of proteins on the arrayed surface is also deleterious for downstream autoantibody binding since it is well known that antibodies tend to bind non-specifically to exposed hydrophobic

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Proteins can be immobilised onto a microarray surface via encapsulation, surface adsorption, covalent attachment or affinity binding (**Figure 4**), which are further described below [67].

Encapsulation of a purified protein on a solid surface involves suspending the protein in a random orientation within a 3D gel pad (e.g. acrylamide or agarose) on an array surface; this approach provides a high capacity for immobilisation and thereby enhances the sensitivity of subsequent assays. A drawback of the technique, however, is that the size of the protein or other ligand applied may restrict diffusion into the gel, resulting in stronger signals at the periphery of the gel pad. This challenge may be surmounted when using different cross-

Immobilisation of purified proteins via noncovalent adsorption is a straightforward, reversible method that involves protein attachment onto a solid support through weak, non-specific interactions, including van der Waals hydrophobic interactions and electrostatic interactions. Commonly used surfaces here include nitrocellulose-coated and amine-terminated glass slides. Although this approach can provide high protein loading onto the surface, the orientation of the immobilised protein cannot be controlled, resulting in variable reaction efficiency, accuracy and reproducibility of the resultant arrays [69]. Furthermore, the underlying sur-

Covalent attachment takes place by chemically cross-linking proteins to the surface through the nucleophilic residues lysine or cysteine. These residues are cross-linked to surface-bound ligands that are terminated with aldehyde, epoxy, or N-hydroxysuccinamide moieties. Irreversible immobilisation of a wide range of proteins to the carrier surfaces are feasible using covalent attachment, but the non-specific modification of surface residues on the arrayed pro-

Affinity capture is a particularly advantageous way to immobilise proteins, since it circumvents many of the limitations of other approaches described above. Typical affinity capture methods include use of biotinylated, hexa-His-tagged, glutathione S transferase tagged or Halo tagged recombinant proteins [1], with orientation of the immobilised protein being controlled via the tag, thereby aiding in preserving the structure and function of the arrayed

Numerous human protein microarray platforms are available today for autoantibody research, including Immunome arrays (Sengenics, Singapore), Nucleic Acid Programmable Arrays (BioDesign Institute, Arizona), Human Protein Atlas Protein Fragment Arrays (SciLifeLab, Sweden), HuProt arrays (CDI Laboratories, USA) and ProtoArrays (ThermoFischer, USA). These various human protein microarray platforms have differing protein content and make

tein carries the risk of altering the activity and folded structure of those proteins [70].

epitopes, giving rise to false positive signals in autoantibody profiling assays.

linkers that can improve the porosity of the gel pads [68].

proteins.

faces tend to be relatively denaturing towards the arrayed proteins [1].

Expression of proteins in yeast is a common alternative to prokaryotic expression systems as it is a well-defined and economical eukaryotic expression system. Commonly used yeast strains include *Saccharomyces cerevisiae* and *Pichia pastoris*, although other yeast strains have also been reported. Proteins expressed using both strains fold efficiently and numerous posttranslational modifications can occur; *P. pastoris* typically gives better protein yields than *S. cerevisiae* [65]. However, a major disadvantage of the yeast expression systems is that they do not mimic protein glycosylation patterns from mammalian cells, with proteins tending to be hyperglycosylated due to the presence of large mannose glycans. Furthermore, lysis conditions for yeast are typically harsh and induce many endogenous proteases, meaning that the extracted recombinant proteins are often significantly proteolysed.

Baculoviruses belong to a diverse group of large double-stranded DNA viruses that infect many different species of insects as their natural hosts but are highly species-specific and are not known to propagate in any non-invertebrate host. Baculoviral expression systems yield good expression levels, especially for intracellular proteins, and typically produce functionally active, recombinant mammalian proteins that are properly folded and oligomerised and which contain correct disulfide bonds, as well as mammalian-like post-translational modifications, including glycosylation, so are both structurally and functionally similar to their native counterparts [65].

Mammalian expression systems are preferred by some researchers as they produce more 'humanised' proteins, with the most biologically-relevant post-translational modifications and native folding. Amongst the most widely used mammalian cells include HeLa, human embryonic kidney-derived (HEK293) epithelial cells, Chinese hamster ovary cells (CHOs) and African green monkey kidney cells (COS). However, mammalian protein expression systems require more demanding culture conditions compared to other systems [65] so are significantly more challenging for high throughput expression purposes.
