Identification Of Differentally Expressed Skin Lesions Proteins Of Psoriasis Vulgaris By Two-dimensional Electrophoresis And Mass Spectrometry

IntroductiomPsoriasis is a common chronic inflammatory skin disease recurrence. Histopathological examination showed thickening of the cuticle, dyskeratosis, thin or granular layer disappears, thickening acanthosis, epidermis angiogenesis. In recent years, the development of proteomics to find the effective ways to sensitivity and specificity of disease. Abnormal protein expression in patients with psoriasis vulgaris usually cause the skin cell death and defense reaction and inflammatory reaction and abnormal psoriasis skin protein expression.In this study, two-dimensional gel electrophoresis (2-DGE) was applied for protein separation, and then identified by MALDI-TOF-TOF-MS as well as bioinformatics technology. Differentially expressed proteins made skin lesions of the comparative analysis of patients with psoriasis vulgaris and normal human tissues, therapeutic value provides thepathogenesis of psoriasis vulgaris..Materials and methodsMaterialsThe specimens of 16 psoriasis vulgaris patients (Male:8; female:8) were collected from Dermatology Department in People's Liberation Army No.202 Hospital. And 20 healthy people (outpatient subjects, male:10; female:10) (psoriasis vulgaris) were recruited as control.The skin layer was collected from skin lesions of psoriasis vulgaris patients and normal control. The area was 0.5 x 0.5cm and was preserved at -70 ? refrigerator. Then, the frozen tissue sections of 16-18 ?m thickness were set on LEICA film slides and then were cut by LEICA laser cutting instrument. The samples from the stratum corneum and dermis tissue were taken. Then, equal amounts of proteins were extracted according to different concentration of proteins. Began two-dimensional gel electrophoresis. Cy3 and Cy5 fluorescence staining was then applied for the dermis group and corneous layer groups in psoriasis vulgaris patients and controls. Then, the image was treated with intensity correction, point detection, background subtraction, homogenization and matching processing by DeCyder 2D 6.5 software. Differentially expressed protein spots choose according to image analysis and cutting.Then, the gel was digested by 20 ?g/L trypsin at 37? overnight. The peptide extraction was dissolved in 50% acetonitrile -0.5% three trifluoroacetic acid. And then, they were mixed with an equal volume of saturated matrix (CCA) and subjected on the stainless steel target instrument, which was further analyzed by mass spectrometry after drying.The methods of assessmentThe peptide mass fingerprint data was searched in the protein sequence database SwissProt and the seach engine was Mascot (http://www.matrixscience.com). Parameter settings are as follows:cysteine as carbamidomethyl-Cys, variable modification as oxidation (M), each peptide allowed 1 incomplete cleavage site peptide fragment with the maximum allowable error of ±100 ppm. Protein matching scores more than 36 scores (P<0.05) mean significant differences. And then the protein names and functions were determined.ResultDimensional gel electrophoresisThe 2-DGE results of stratum comeum and dermis from psoriasis vulgaris patients and normal human was analyzed using DeCyder 2D 6.5 image analysis software. The protein spots showed reproducibility and stability very well. According to Figure 2, the differential protein spots were 1969±21 and 1928±49, respectively. One piece of gel was set as control, with the average matching rate of 78.2% and 76.3%. If the ratio of the differentially expressed proteins were greater than 2 and the same changes appear in three pieces of gels, they were treated as the differentially expressed proteins.MS identification resultsIn this study, the proteins from the stratum corneum and dermis layer of skin lesions of psoriasis vulgaris patients and normal human were selected as the research object. Based on the MANLDI MS and bioinformatics analysis of differentially expressed proteins, fourteen proteins were identified. After recognition, these proteins may be classified into the following collections(Table 1):1. Type 1 keratin cytoskeletal proteins (K1C10, K1C14, K1C15, K1C16),2. Type 2 keratin cytoskeletal protein (K2C1),3. actin (ARP3, ACTA, ACTBM),4. heat shock protein 4 (PHB, HSP ? 1, CH60),5. centrosome protein (CP135), membrane associated protein (ANXA4, ANXA5). Differential protein spots in the cuticle of a patient with psoriasis vulgaris.In the cuticles of psoriasis vulgaris patients, K2C1, K1C10, K1C16, K1C14, CH60, PHB, ARP3, ACTBM, ACTA, ANAX5, CP135, and PHB were highly expressed, while K1C15, ANAX4, and HSP?1 were expressed lower. Proteins differentially expressed in patients with psoriasis vulgaris leather.In the dermis of psoriasis vulgaris patients, K1C16, CP135, and ACTA were expressed lower, among which K1C16, CP135, and ACTA expression were higher in psoriasis vulgaris horny layer and lower in dermis.ConclusionDifferential expressed proteins in psoriasis vulgaris lesions and normal tissue play key roles in the pathology.Used as a potential diagnosis flag, clarify the pathogenesis of these proteins explain psoriasis vulgaris has important significance.