Proteomic anatomy of human skin.

The extracellular matrix is composed of a variety of proteins which are essential for growth, wound healing, and fibrosis. It provides both structural support as well as contributing to the regulation of the local microenvironment. To further characterize the molecular composition of human skin we have undertaken a proteomic approach to identify proteins in three skin regions from two locations. Using laser microdissection, extracellular matrix was obtained from three distinct regions (basement membrane, papillary dermis, and reticular dermis) of formalin-fixed, paraffin embedded tissue from normal human leg and breast skin. The proteome of these regions was determined by mass spectrometric analysis. This study provides a relative quantitative assessment, including protein composition and relative abundance, of the proteins found in different skin regions giving rise to a "proteomic anatomy" of skin. Our findings indicate that there was little difference detected in the subproteomes of the dermal and papillary regions and little difference between the proteomes of leg skin compared to breast skin. One finding of interest is the identification of tenascin-X only in the breast dermis and serum amyloid P-component in the leg dermis. The results of this proteomic analysis corroborate much of the information on the protein composition identified by other methodologies found in the literature but provide additional insight as to localization of skin proteins in the various regions of skin. One potential outcome of this study is that identification of a more global proteomic composition in normal skin may serve as the basis for characterizing and comparing the skin proteomes from a variety of disease states, which may lead to a more complete understanding of the pathology of the disease as well as new therapeutic treatments. BIOLOGICAL SIGNIFICANCE: This investigation underscores the power of proteomics to bring semiquantitative, non-presumptive molecular characterization to the field of histological anatomy. Traditionally, anatomy relied on visual or histochemical structural characterization which generally involved some level of understanding of the area of interest. With the advent of laser microdissection or laser capture microscopy localization of anatomical structures of interest can be correlated to molecular composition by virtue of mass spectrometric determination of the proteome of that structure. One potential outcome of this study is that identification of a more global proteomic composition in normal skin may serve as the basis for characterizing and comparing the skin proteomes from a variety of disease states, which may lead to a more complete understanding of the pathology of the disease as well as new therapeutic treatments.

Evaluation of molecular markers of mesenchymal phenotype in melanoma.

The epithelial to mesenchymal transition is a developmental process allowing epithelial cells to dedifferentiate into cells displaying mesenchymal phenotypes. The pathological role of epithelial to mesenchymal transition has been implicated in invasion and metastasis for numerous carcinomas, yet limited data exist addressing whether mesenchymal transition (MT) occurs in malignant melanoma cells. Our group developed an in-vitro three-dimensional culture system to address MT in melanoma cells upon transforming growth factor-ß/ tumor necrosis factor-α treatment. Loss of E-cadherin is one of the best indicators of MT in epithelial cells. Not surprisingly, E-cadherin was expressed in only three of 12 (25%) melanoma cell lines and all three mesenchymal proteins, N-cadherin, vimentin, and fibronectin, were expressed by seven (58%) melanoma cell lines. However, after cytokine treatment, two or more mesenchymal proteins were elevated in nine (75%) melanoma cell lines. Data support the transforming growth factor-ß production by melanoma cells which may induce/support MT. Evaluation of E-cadherin, N-cadherin, and Snail expression in melanoma tissue samples are consistent with an inverse coupling of E-cadherin and N-cadherin expression, however, there are also examples suggesting a more complex control of their expression. These results indicate that malignant melanoma cell lines are susceptible to MT after cytokine treatment and highlight the importance of understanding the effects of cytokines on melanoma to undergo MT.

The utility of ETD mass spectrometry in proteomic analysis.

Mass spectrometry has played an integral role in the identification of proteins and their post-translational modifications (PTM). However, analysis of some PTMs, such as phosphorylation, sulfonation, and glycosylation, is difficult with collision-activated dissociation (CAD) since the modification is labile and preferentially lost over peptide backbone fragmentation, resulting in little to no peptide sequence information. The presence of multiple basic residues also makes peptides exceptionally difficult to sequence by conventional CAD mass spectrometry. Here we review the utility of electron transfer dissociation (ETD) mass spectrometry for sequence analysis of post-translationally modified and/or highly basic peptides. Phosphorylated, sulfonated, glycosylated, nitrosylated, disulfide bonded, methylated, acetylated, and highly basic peptides have been analyzed by CAD and ETD mass spectrometry. CAD fragmentation typically produced spectra showing limited peptide backbone fragmentation. However, when these peptides were fragmented using ETD, peptide backbone fragmentation produced a complete or almost complete series of ions and thus extensive peptide sequence information. In addition, labile PTMs remained intact. These examples illustrate the utility of ETD as an advantageous tool in proteomic research by readily identifying peptides resistant to analysis by CAD. A further benefit is the ability to analyze larger, non-tryptic peptides, allowing for the detection of multiple PTMs within the context of one another.

Identification of class I MHC-associated phosphopeptides as targets for cancer immunotherapy.

Alterations in phosphorylation of cellular proteins are a hallmark of malignant transformation. Degradation of these phosphoproteins could generate cancer-specific class I MHC-associated phosphopeptides recognizable by CD8+ T lymphocytes. In a comparative analysis of phosphopeptides presented on the surface of melanoma, ovarian carcinoma, and B lymphoblastoid cells, we find 5 of 36 that are restricted to the solid tumors and common to both cancers. Differential presentation of these peptides can result from differential phosphorylation of the source proteins. Recognition of the peptides on cancer cells by phosphopeptide-specific CD8+ T lymphocytes validates the potential of these phosphopeptides as immunotherapeutic targets.