Comparative Proteomic Profiling of Murine Skin Chun-Ming Huang, K. Wade Foster, Tivanka DeSilva, JianFeng Zhang, Zhongkai Shi, Nabiha Yusuf, Kent R. Van Kampen, Craig A. Elmets, De-chu C. Tang  Journal of Investigative Dermatology  Volume 121, Issue 1, Pages 51-64 (July 2003) DOI: 10.1046/j.1523-1747.2003.12327.x Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Two-dimensional proteomic profiles of murine ventral skin. Samples containing 300 μg of protein from BALB/c (A, B) and C3H/HeN (C) mice were subjected to IEF in linear gradient Immobiline Dry-Strips of pH 3–10 (A) or pH 4–7 (B, C), separated according to mass via SDS polyacrylamide gel electrophoresis, and then silver stained. Fifty-two protein spots from BALB/c skin (circled) and seven protein spots from C3H/HeN skin (1, 28, 36, 46, and 53–55) were subsequently excised and analyzed by in-gel trypsin digestion followed by MALDI-TOF MS. Peptide mass fingerprint spectra were analyzed, interpreted, and matched to SWISS-PROT database entries using the Mascot database searching algorithm (Table I). Proteins identified from BALB/c skin were matched to spots present in the C3H/HeN proteome using PDQuest software. Predictions were confirmed by the analysis of selected spots in the C3H/HeN proteome (bold) using MALDI-TOF MS. All numbered spots except 11, 12, and 42 in panel B are present in panel A. Protein spots 53–55 are present only in C3H/HeN mice. Asterisks indicate strain-specific protein expression. Journal of Investigative Dermatology 2003 121, 51-64DOI: (10.1046/j.1523-1747.2003.12327.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Peptide mass fingerprints of cutaneous proteins. Fingerprint mass spectra and the predicted peptide fragments corresponding to observed m/z values for (A) HSP27, spot 1; (B) Cu/Zn superoxide dismutase, spot 18; (C) ER60, spot 26; and (D) galectin-7, spot 19, are shown. The tryptic autodigestive peak at m/z=2164.05 (asterisk) served as an internal calibration standard. Journal of Investigative Dermatology 2003 121, 51-64DOI: (10.1046/j.1523-1747.2003.12327.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Identification of proteins with predominantly epidermal or subepidermal expression. Murine skin was treated with 0.5 M ammonium thiocyanate to induce separation, and 300 μg protein derived from epidermis (A) or subepidermal tissues (B) was subjected to IEF in linear gradient Immobiline Dry-Strips of pH 4–7 and then separated according to mass via SDS polyacrylamide gel electrophoresis. Gels were subsequently silver stained, and proteins were quantified using PDQuest software. Identities of proteins with increased expression levels in the subepidermal tissues (squares) or epidermis (circles) were confirmed by MALDI-TOF MS. Differences in protein expression levels were greater than 1.5-fold in three independent experiments and were found to be statistically significant (p<0.01) using a Student's t test. Journal of Investigative Dermatology 2003 121, 51-64DOI: (10.1046/j.1523-1747.2003.12327.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Prohibitin expression in murine skin. Fingerprint mass spectra and the predicted peptide fragments corresponding to observed m/z values for prohibitin (spot 17). The tryptic autodigestive peak at m/z=2164.05 is indicated by an asterisk (A). Epidermal (E) and subepidermal (SE) prohibitin protein expression from silver-stained two-dimensional gels (left). PDQuest software was used to quantify protein abundance (right) (B). RT-PCR was performed on total RNA isolated from epidermal (E) and subepidermal (SE) tissues using primers specific for prohibitin and APRT (left). Lanes 1–4, 10-fold serial dilutions of cDNA template; lane 5, no template. Epidermal and subepidermal prohibitin mRNA expression was normalized against the respective expression levels of APRT mRNA and compared (right) (C). Statistical analysis of data from three independent experiments was performed using a Student's t test, and differences reaching statistical significance are indicated by an asterisk (p≤0.01). Error bars denote mean ± SD. Journal of Investigative Dermatology 2003 121, 51-64DOI: (10.1046/j.1523-1747.2003.12327.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Analysis of HSP27 expression in murine keratinocytes, antigen-presenting cells, and cutaneous T cells. (A) Detection of HSP27 in mouse skin (green). (B) Control, no anti-HSP27 primary antibody. (C) Superimposed image of K10-positive (red), HSP27-positive (green), and double-positive (yellow, arrows) cells. (D) Superimposed image of K14-positive (red), HSP27-positive (green), and double-positive (yellow, arrows) cells. (E) Superimposed image of MHC-class-II-positive (red) and HSP27-positive (green) cells. (F) Superimposed image of CD3-positive (red) and HSP27-positive (green) cells. All sections were counterstained with Hoechst 33258. Scale bars: 20 μm. Journal of Investigative Dermatology 2003 121, 51-64DOI: (10.1046/j.1523-1747.2003.12327.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Changes in the expression of cutaneous molecular chaperones following heat and cold shock. Protein aliquots (300 μg) derived from murine abdominal skin exposed to temperatures of 0°C (hatched), 25°C (white), or 45°C (black) were separated by 2-DE. Following silver-staining, levels of HSP27, ER60, GRP78, HSP70, and HSP60 were quantified using PDQuest software (A). Differences are expressed as a percentage of the control (B). Statistical analysis of data from three independent experiments was performed using Student's t test. Differences from the control group reaching statistical significance are indicated by a single asterisk (p<0.05) or double asterisk (p<0.01). Error bars denote mean±SD. Changes in HSP27, HSP70, and HSP60 protein expression were confirmedby western blot analysis (C). Journal of Investigative Dermatology 2003 121, 51-64DOI: (10.1046/j.1523-1747.2003.12327.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions