Proteomic analysis of balding and non-balding mesenchyme-derived dermal papilla cells from androgenetic alopecia patients using on-line two-dimensional reversed phase-reversed phase LC-MS/MS.

Androgenetic alopecia (AGA) is the most common and progressive disorder of hair loss with psychological effects. However, the exact mechanisms of baldness have not yet been elucidated. Until now, there has been no report using current proteomic approaches to examine balding and non-balding mesenchyme-derived DPCs simultaneously. To achieve the goal of identifying differentially expressed proteins in balding DPCs compared to non-balding DPCs, we present a strategy combining gel-assisted digestion and automatic on-line two-dimensional reversed phase-reversed phase (2D RP-RP) LC-MS/MS with informatics-assisted label-free protein quantitation. This strategy efficiently quantified the proteins of balding and non-balding DPCs from patients, and 128 up-regulated and 12 down-regulated proteins among 690 distinct proteins were identified in balding DPCs compared to non-balding DPCs. Up-regulated proteins belonging to these pathways in the balding DPCs, including argininosuccinate synthase 1 (ASS1), phosphoribosylaminoimidazole carboxylase (PAICS; ADE2), cytoskeleton-associated protein 4 (CKAP4), gelsolin (GSN), Ras GTPase-activating-like protein (IQGAP1), and S-phase kinase-associated protein 1 (SKP1), were confirmed by Western blot analysis. Gelsolin (GSN), Ras GTPase-activating-like protein (IQGAP1), plectin-1 (PLEC1), and nonerythroid α-spectrin (SPTAN1) were confirmed by immunofluorescence analysis. This proteomic approach to DPCs should help in understanding the pathogenesis of AGA. BIOLOGICAL SIGNIFICANCE: The results demonstrated the first deep proteomic approach to mesenchyme-derived dermal papilla cells (DPCs), which are a useful model system to investigate the mechanisms of baldness, from human hair and also identified differentially expressed proteins in balding DPCs compared to non-balding DPCs. This study should help in understanding the pathogenesis of androgenetic alopecia (AGA) and furthermore some proteins differentially expressed could be studied as therapeutic targets for AGA. Crown