Proteomic Studies of Psoriasis

Abstract

In this review paper, we discuss the contribution of proteomic studies to the discovery of disease-specific biomarkers to monitor the disease and evaluate available treatment options for psoriasis. Psoriasis is one of the most prevalent skin disorders driven by a Th17-specific immune response. Although potential patients have a genetic predisposition to psoriasis, the etiology of the disease remains unknown. During the last two decades, proteomics became deeply integrated with psoriatic research. The data obtained in proteomic studies facilitated the discovery of novel mechanisms and the verification of many experimental hypotheses of the disease pathogenesis. The detailed data analysis revealed multiple differentially expressed proteins and significant changes in proteome associated with the disease and drug efficacy. In this respect, there is a need for proteomic studies to characterize the role of the disease-specific biomarkers in the pathogenesis of psoriasis, develop clinical applications to choose the most efficient treatment options and monitor the therapeutic response.

Keywords: LC-MS/MS; SOMAscan™; biomarkers; comorbidities; mass spectrometry; predisposition; proximity extension assay; psoriasis; risk factors.

Figure 1

2D-gel electrophoresis followed by mass spectrometry. The experiment starts from electrophoretic separation of protein samples in two directions, by isoelectric point and by molecular weight (M_w). Digestion in- or out-of-gel, mass spectrometry, and data analysis follow electrophoresis.

Figure 2

Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The experiment begins with a protein separation by liquid chromatography (LC) followed by ionization and mass spectrometry analysis. Triple quadrupole mass spectrometer comprises of three quadrupoles Q₁, Q₂, and Q₃. Quadrupoles Q₁ and Q₃ serve as mass filters. The middle quadrupole Q₂ functions as a collision cell. The specific precursor ions isolated by Q₁ and accumulated in Q₂ collide with neutral gas molecules, such as nitrogen generating the product ions. The product ions reach the third quadrupole assembly Q₃. The quadrupole Q₁ allows ions with a specific m/z ratio range to pass to the detector (D). P—pump; C –column; ES—electrospray, a liquid-to-aerosol converting device.

Figure 3

iTRAQ workflow. Several samples are separately digested and labeled with iTRAQ tags of different molecular weights and pulled. The labeled peptides are separated by HPLC followed by mass spectrometry. During mass spectrometry, tags become released from the peptide. The ratio of the iTRAQ ions is used to assess the sample-specific content of a particular peptide.

Figure 4

The regulation of estrogen-responsive proteins in lesional skin of female psoriasis patients. The signs ↓ and ↑ indicate that the protein expression is increased and decreased, respectively. The sign ⇆ indicates that chemical reaction between HCO₃⁻ and can be reversed [48].

Figure 5

The principle of the SOMAscan. (A). Aptamers used in the study interact with a specific protein (target). Each aptamer contains three parts: a single-stranded DNA made of both canonical and modified deoxyribonucleotides (light blue), fluorophore (green), a UV-sensitive spacer (red), and biotin (grey). (B). The specific interaction of an aptamer with a target protein. The nucleotides directly interacting with a protein are shown in red and the others—in dark blue. A SOMAscan includes several steps. (C). Incubation of aptamers with streptavidin beads (Step 1). Because the aptamers contain biotin, they bind to the beads via biotin. (D). Incubation of streptavidin-coupled aptamers with the desired protein sample (Step 2). As protein samples used in the experiment presumably contain target proteins, aptamers specifically interact with their target protein in the 1:1 ratio. (E). Biotinylation of the target proteins with biotin, bt (Step 3). (F). Photosensitive cleavage of the spacers from aptamers followed by a separation of aptamers from the beads (Step 4). (G). The released complexes of aptamers and biotinylated proteins become recaptured by streptavidin beads (Step 5). As target proteins are biotinylated, they interact with streptavidin beads via biotin. (H). Dissociation of aptamers from their target proteins (Step 6). (I). Hybridization of aptamers with the probes of complementary single-stranded DNA attached to a slide (Step 7). Since each aptamer contains a fluorophore, the hybrid dsDNA molecules emit a fluorescence. The intensity of emitted light is proportional to the level of the corresponding target protein since it interacted with aptamer in the 1:1 ratio.

Figure 6

The Proximity Extension Assay. The experiment includes several steps. (A). Immunoassay (Step 1). The pairs of specific antibodies conjugated with ssDNA oligonucleotides specifically interact with target proteins. As both ssDNA contain short complementary sequences, they interact and form a duplex in the middle. (B). Extension of ssDNA (Step 2). The duplex serves as a set of primers to DNA polymerase that extends ssDNA to dsDNA. (C). A cleavage of dsDNA (Step 3). Cutting dsDNA from the antibodies produces an oligonucleotide. Each oligonucleotide contains a unique sequence that serves as a barcode to identify the target protein. It also has two short adapter sequences at the ends. (D). Preparation for the sequencing of the barcodes (Step 4). The obtained double-stranded oligonucleotides are denatured and interact with immobilized probes complementary to the adapters. Then, DNA polymerase amplifies DNA using the probes as primers. (E). Clusterization of DNA and sequencing the barcodes (Step 5). As the concentration of DNA is relatively low, the amplified molecules form clusters. Each cluster originates from a single DNA molecule and represents a specific barcode. As the “clusters” have to be homogenous on their composition, the dsDNA is denatured to wash out the disconnected ssDNA. In addition, DNA attached to the slide through one of the adapters (e.g., adapter 2) becomes cut and removed. The following sequencing of the DNA identifies and quantifies the barcodes. The numbers of identified barcodes are proportional to the levels of the corresponding target proteins since the single interaction of specific antibodies to a target protein produces only one barcode.