Use Of The Label-free LC-MS/MS For Non-invasive Quantitative Proteomic Analysis Of Skin Barrier Related Proteins In The Patients With Psoriasis Vulgaris

Study background Psoriasis is generally considered as an immune-induced disease,and many studies recently have found that psoriasis might be driven by the major defect of epidermal skin barrier function.The skin barrier function typically refers to that of the epidermis,particularly the stratum corneum,and the proteins in the stratum corneum are the important biochemical components of skin barrier structure.Any abnormal expression in the proteins will affect the skin barrier functions of the patients with psoriasis.Keratin(KRT)is the main structural protein of epidermal cells,fibriform,forming the compact supporting structure of stratum corneum comprised of keratin fiber bundles with filaggrin(FLG).Filaggrin(FLG),together with loricrin(LOR),involucrin(IVL),trichohyalin,small prolin-rich proteins(SPRRs)and other proteins,forms the cornified envelope(CE)under the cross-linking of transglutaminase(TGs).CE plays an important role in fighting against external mechanical tension,preventing the pathogen invasion and maintaining the water balance inside and outside the stratum corneum,and effectively prevents the external hazardous substances from skin invasion,acting as the foundation of defense barrier for the stratum corneum.Filaggrin located in the granular layer is transformed into biologically active filaggrin under the effect of cysteiyl aspartate specific proteinase-14(Caspase-14)and concentrates on the stratum corneum;filaggrin is further degraded into free amino acids under the effect of various proteolytic enzymes in the stratum corneum.For example,urocanic acid,pyrrolidone-5-carboxylated acid,ornithine,citrulline and aspartic acid are hydrophilic,forming the key components of natural moisturizing factor,playing an important role in maintaining the hydrating and moisturizing functions of stratum corneum.In addition to the above keratin and keratin-related proteins,S100 A protein and tight junction protein(TJ)are also found to play an important role in maintaining the skin barrier function.The association of the above proteins closely related to the skin barrier function of stratum corneum,i.e.KRT,FLG,LOR,IVL,S100 A protein,SPRRs and TGs,with psoriasis has also become a research focus.At present,the research technique in this field is skin biopsy in combination with immunohistochemistry.Study objective To explore the difference in the expression of skin barrier related proteins in the stratum corneum of the patients with psoriasis vulgaris and the normal subjects by collecting the skin samples from the stratum corneum of the normal subjects and the patients with psoriasis vulgaris in a non-invasive manner and by making label-free quantitative analysis on the proteins in the stratum corneum with the liquid chromatography with tandem mass spectrometry(LC-MS/MS),so as to provide a good methodology for more deeply investigating the skin barrier related proteins in the patients with psoriasis in the future.Materials and methods The skin was sampled from the skin lesion area at the forearm flexor side of patients with psoriasis vulgaris in the test group with a 3L medical pressure-sensitive adhesive tape,and likewise,the skin was sampled from the forearm flexor side of normal subjects in the control group with a 3L medical pressure-sensitive adhesive tape.The proteins on the tapes of the two groups were collected,extracted and enzyme-hydrolyzed for LC-MS/MS analysis of the trypsin-degraded peptide mixture by using a high-resolution mass spectrometer.Finally,Max Quant was used for search of label-free database,and Max LFQ was used to calculate the final result of each sample.Results: In this study,1102 proteins were detected in the patients with psoriasis,and106 proteins were detected in the normal subjects.Through statistical analysis of the peak area value of each protein in the normal group and the psoriasis vulgaris group,we found that there were 41 protein peak area values closely related to the skin barrier function,there was significant difference between the two groups,the difference was statistically significant.Conclusion: There is significant difference in the expression of skin barrier related proteins KTR1,KRT2,KRT5,KRT6 A,KRT6B,KRT8,KRT9,KRT10,KRT14,KRT16,KRT17,KRT72,SPRR1 A,SPRR2D,SPRR2 B,SPRR2F,SPRR1 B,SPRR2E,SPRR2 A,IVL,FLG2,TGM3,TGM1,TGM5,LCE3 E,LCE3D,LOR,FLG,Caspase-14,S100A8,S100A9,S100A7,S100A7 A,S100A3,S100A11,S100A16,S100A2,desmoplakin(DSP),desmoglein1(DSG1),desmoglein3(DSG3),TPJ1 and TPJ2 in the patients with psoriasis vulgaris compared to normal subjects.The difference in the expression of these proteins affects the differentiation and proliferation of keratinocytes in the patients with psoriasis vulgaris as well as the epidermal barrier function in the patients with psoriasis vulgaris,and meanwhile,the related inflammatory factors of psoriasis vulgaris also affect the expression of these skin barrier related proteins.The non-invasive sampling technique used in this study is different from the common invasive skin biopsy sampling technique,and is characterized by non-invasiveness,high sensitivity and high accuracy in combination with the label-free up-to-date proteomic technique LC-MS/MS,providing a good methodology for deepening and expanding the studies associated with the skin barrier proteins and psoriasis vulgaris in the future.