Dihydrolipoyl dehydrogenase as a potential UVB target in skin epidermis; using an integrated approach of label-free quantitative proteomics and targeted metabolite analysis Eunjung Moon a , Hye Min Park b , Choong Hwan Lee b , Seon-Gil Do c , Jong-Moon Park a , Na-Young Han a , Moon Ho Do a , Jong Ha Lee a , Hookeun Lee a, d, e , ⁎ , Sun Yeou Kim a, d, ⁎⁎ a College of Pharmacy, Gachon University, Incheon, Republic of Korea b Department of Bioscience and Biotechnology, Konkuk University, Seoul, Republic of Korea c Life Science Research Institute, Univera, Inc., Seoul, Republic of Korea d Gachon Medical Research Institute, Gil Medical Center, Incheon, Republic of Korea e Lee Gil Ya Cancer and Diabetes Institute, Gachon University, Incheon, Republic of Korea ARTICLE INFO ABSTRACT Article history: Received 10 May 2014 Accepted 12 December 2014 Photodamage is extrinsically induced by overexposure to ultraviolet (UV) radiation, and it increases the risk of various skin disorders. Therefore, discovery of novel biomarkers of photodamage is important. In this study, using LC–MS/MS analysis of epidermis from UVB-irradiated hairless mice, we identified 57 proteins whose levels changed after UVB exposure, and selected 7 proteins related to the tricarboxylic acid (TCA) cycle through pathway analysis. Dihydrolipoyl dehydrogenase (DLD) was the only TCA cycle-associated protein that showed a decreased expression after the UVB exposure. We also performed targeted analysis to detect intermediates and products of the TCA cycle using GC–TOF-MS. Interestingly, malic acid and fumaric acid levels significantly decreased in the UVB-treated group. Our results demonstrate that DLD and its associated metabolites, malic acid and fumaric acid, may be candidate biomarkers of UVB-induced skin photoaging. Additionally, we showed that Aloe vera, a natural skin moisturizer, regulated DLD, malic acid and fumaric acid levels in UVB-exposed epidermis. Our strategy to integrate the proteome and targeted metabolite to detect novel UVB targets will lead to a better understanding of skin Keywords: Integration of proteomics and targeted metabolite analysis UVB targets Photoaging Epidermis Dihydrolipoyl dehydrogenase Aloe vera JOURNAL OF PROTEOMICS 117 (2015) 70 – 85 Abbreviations: DLD, dihydrolipoyl dehydrogenase; DMEM, Dulbecco's modified Eagle's medium; FASP, filter-aided sample preparation; FBS, fetal bovine serum; HBSS, Hank's balanced salt solution; IPA, ingenuity pathway analysis; IVN, involucrin; MMP-1, matrix metalloproteinase-1; PC-1, procollagen type 1; PS, penicillin–streptomycin; ROS, reactive oxygen species; SA-β-Gal, senescence- associated β-galactosidase; SDS, sodium dodecyl sulfate; SNR, signal-to-noise ratio; TBST, Tris-buffered saline-T; TCA cycle, tricarboxylic acid cycle; UV, ultraviolet. ⁎ Correspondence to: H. Lee, Gachon Medical Research Institute, Gil Medical Center, Incheon, Republic of Korea. Tel.: + 82 32 899 6584; fax:+ 82 32 899 6519. ⁎⁎ Correspondence to: S.Y. Kim, College of Pharmacy, Gachon University, Incheon, Republic of Korea. Tel.: +82 32 899 6411; fax: +82 32 899 6591. E-mail addresses: jubbi@hanmail.net (E. Moon), ramgee@naver.com (H.M. Park), chlee123@konkuk.ac.kr (C.H. Lee), sgildo@univera.com (S.-G. Do), bio4647@naver.com (J.-M. Park), ddasehan@nate.com (N.-Y. Han), dvmoono@naver.com (M.H. Do), whdgkfl@naver.com (J.H. Lee), hklee@gachon.ac.kr (H. Lee), sunnykim@gachon.ac.kr (S.Y. Kim). http://dx.doi.org/10.1016/j.jprot.2014.12.016 1874-3919/© 2015 Published by Elsevier B.V. Available online at www.sciencedirect.com ScienceDirect www.elsevier.com/locate/jprot