Interactome Mapping: Using Protein Microarray Technology to Reconstruct Diverse Protein Networks

Uzoma, Ijeoma; Zhu, Heng

In many autoimmune diseases, there is an unmet clinical need for cost-effective and accurate diagnostic methods. Improving upon the current standard requires discovery and characterization of reliable autoantigens coupled with sensitive and reproducible assays. Take autoimmune hepatitis (AIH) as an example: AIH is a chronic necroinflammatory disease of human liver with little known etiology. Detection of non-organ-specific and liver-related autoantibodies using immunoserological approaches has been widely used for diagnosis and prognosis [57] . However, these traditional autoantigens, such as anti-smooth muscle autoantibodies (SMA) and anti-antinuclear autoantibodies (ANA) are often mixtures of complex biological materials. Unambiguous and accurate detection of the disease demands identification and characterization of these autoantigens. Therefore, Song et al. fabricated a human protein microarray of 5011 non-redundant proteins that were expressed and purified as GST fusions in yeast [25] . There are several advantages associated with producing human proteins in yeast rather than bacteria: (1) higher solubility, (2) higher yields of large proteins (e.g., >50 kD), (3) better preserved conformation of proteins and (4) less immunogenicity of proteins when produced in yeast than in Escherichia coli [7, 12, 17] . However, unlike a viral or bacterial protein microarray, a significant obstacle to the use of a human protein microarray of high content is the high cost. For example, cost for a human protein array of 9000 proteins can exceed $1000 per array. In order to reduce the cost, Song et al. developed a two-phase strategy to identify new biomarkers in AIH. Phase I is designed for rapid selection of candidate biomarkers, which are then validated in Phase II (Figure 1) . In Phase I, serum samples from 22 AIH patients and 30 healthy controls were selected and individually used to probe the human protein microarrays at a 1000-fold dilution, followed by detection of bound human autoantibodies using a Cy-5-conjugated anti-human IgG antibody. Statistical analysis revealed 11 candidate autoantigens. To validate these candidates and to avoid a potential overfitting problem (see below), which is especially likely when dealing with a small sample size, the 11 proteins and 3 positive controls were re-purified to build a large number of low-cost small arrays for Phase II validation. These arrays were then sequentially probed with serum samples used in Phase I and serum samples obtained from an additional 52 AIH, 50 primary biliary cirrhosis (PBC), 43 hepatitis B virus (HBV), 41 hepatitis C virus (HCV), 11 system lupus erythematosus (SLE) and 11 primary Sjo¨gren's syndrome (pSS) patients. As negative controls, they also included 26 serum samples from patients suffering from other types of severe diseases and 50 samples from healthy subjects. Three new antigens, RPS20, Alb2-like and dUTPase, were identified as highly AIH-specific biomarkers with sensitivity of 47.5%, 45 .5% and 22.7%, respectively, which were further validated with additional AIH samples in a double-blind design. Finally, they demonstrated that these new biomarkers could be readily applied to ELISA-based assays for clinical diagnosis and prognosis [25] . Figure 1 Scheme of the two-phase strategy for biomarker identification in human autoimmune diseases taking AIH as example In Phase I, a small cohort is used to rapidly identify a group of candidate biomarkers via serum profiling assays o