Mass spectrometry data from proteomic analysis of human skin keratins after exposure to UV radiation

A mass spectrometry (MS)-based proteomic methodology was employed to monitor oxidative modifications in keratins, the main constituents of human skin (“Non-invasive proteomic analysis of human skin keratins: screening of methionine oxidation in keratins by mass spectrometry” [1], “UV irradiation-induced methionine oxidation in human skin keratins: mass spectrometry-based non-invasive proteomic analysis” [2]). Human skin proteins were obtained non-invasively by tape stripping and solubilized in sodium dodecyl sulfate (SDS) buffer, followed by purification and digestion using the filter-aided sample preparation method. The tryptic peptides were then analyzed by liquid chromatography (LC)/electrospray ionization (ESI)-MS, tandem MS (MS/MS), and LC/ESI-selected reaction monitoring (SRM)/MS. The MS/MS data were generated to confirm amino acid sequences and oxidation sites of tryptic peptides D290VDGAYMTK298 (P1) and N258MQDMVEDYR267 (P2), which contain the most susceptible oxidation sites (Met259, Met262, and Met296 in K1 keratin) upon UVA irradiation [2]. Subsequently, quantitative determination of the relative oxidation levels of P1 and P1 [2] was achieved by LC/ESI-SRM/MS analyses of P1 and P2 together with their oxidized forms after exposure to UVA radiation or treatment with hydrogen peroxide (H2O2).

forms after exposure to UVA radiation or treatment with hydrogen peroxide (H 2 O 2 ).
& 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Subject area
Toxicology and pharmacological science More specific subject area

Proteomics
Type of data MS/MS spectra, LC/ESI-SRM/MS chromatograms How data was acquired Mass spectrometry using a API2000 triple quadrupole mass spectrometer (ABsciex, Framingham, MA)

Data format
Analyzed by Analyst s Version 1.5.2.

Experimental factors
Human skin proteins were obtained non-invasively by tape stripping and solubilized in SDS buffer, followed by purification and digestion using the filteraided sample preparation method.

Experimental features
Human skin tryptic peptides were analyzed by LC/ESI-MS, MS/MS, and LC/ESI-SRM/MS. Data source location Department of Bio-analytical Chemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi 980-578, Japan Data accessibility Data is with this article.

Value of the data
The MS/MS data confirmed the identities of tryptic keratin peptides carrying the most susceptible oxidation sites.
The SRM/MS data revealed a time-dependent increase in the relative oxidation levels of target keratin peptides upon UVA irradiation and H 2 O 2 treatment.
Difference in the oxidation sites between UV and H 2 O 2 treatment was found. The relative oxidation levels reported could be applied to the assessment of oxidative stress levels in skin after exposure to sunlight.
The relative methionine (Met) oxidation levels in keratins could be used as dosimeters of skin damage.

Data
Matrix-assisted laser desorption ionization/time of flight-MS analyses of UVA-irradiated human skin proteins revealed Met-containing tryptic peptides, D 290 VDGAYMTK 298 (P1) and N 258 MQDMVEDYR 267 (P2), as potential biomarkers of oxidative skin damage [2]. Amino acid sequences and oxidation sites of P1 and P2 were confirmed by the MS/MS analysis and comparisons to their authentic standards (Fig. 1). The UVinduced oxidative susceptibility of peptides P1 and P2 was assessed via irradiating human tape-stripped skin proteins with UVA for 0-48 h, followed by LC/ESI-SRM/MS analysis (Fig. 2). The extent of UVAmediated oxidation of target peptides P1 and P2 was compared with that induced by H 2 O 2 (Fig. 3).

Isolation and solubilization of human skin proteins
Human skin proteins were obtained by tape stripping the outermost layer of the skin with adhesive skin tapes. The tape was attached to the upper arm, gently pressed three times with a finger, and detached. To avoid contamination by surface lipids, the upper arm skin was cleaned with ethanol before the procedure and the first tape strip was discarded. After the second tape stripping, onefourth (6.25 Â 25 mm 2 ) of the tape was placed into a 2 mL microcentrifuge tube containing 200 μL of SDS buffer (0.1% SDS, 0.15 M NaCl, and 2 mM butylated hydroxytoluene (BHT) in 50 mM sodium phosphate buffer, pH 7.0) with the adhesive side facing inward. Human skin proteins on the gluey surface of the skin tape were then solubilized in the buffer by scraping them for 4 min using a grinding plastic pestle followed by sonication for 1 min. The sonicated human skin protein mixture was centrifuged at 11,300 g for 10 min at 4°C.     filtration unit and centrifuged for 12 min at 11,300 g. The mixture remaining in the filter unit was diluted with 100 μL of RCM buffer and centrifuged (11,300 g for 12 min). After centrifugation, the mixture was diluted with 100 μL of RCM buffer, incubated in darkness at room temperature for 45 min with 25 μL of iodoacetamide (110 mM) in RCM buffer, and centrifuged for 12 min. The mixture in the filter unit was diluted with 100 μL of 100 mM NH 4 HCO 3 containing 2 mM BHT and centrifuged for 12 min at 11,300 g. This step was repeated three times. The filter unit containing human skin protein mixture was transferred to a new collection tube and added to 100 μL of 100 mM NH 4 HCO 3 containing 2 mM BHT and 0.75 μg of trypsin. Following overnight incubation at 37°C, peptides were collected by centrifugation of the filter unit for 12 min at 11,300 g. The filtrate was evaporated to dryness under a nitrogen stream and redissolved in 30 μL of 5% aqueous acetonitrile for LC/ESI-MS analysis.