Mass spectrometry has played an integral role in the identification of proteins and their posttranslational 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 posttranslationally 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.

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.tandem mass spectrometry ͉ immobilized metal-affinity chromatography

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Epithelial to mesenchymal transition is a developmental process allowing epithelial cells to dedifferentiate into cells displaying mesenchymal phenotypes. The pathological role of EMT 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 3D culture system to address MT in melanoma cells upon TGF-β/TNF-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 twelve (25%) melanoma cell lines and all three mesenchymal proteins, N-cadherin, vimentin, and fibronectin, were expressed by seven (58%) lines. However, following cytokine treatment, two or more mesenchymal proteins were elevated in nine (75%) lines. Data support the TGF-β 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 following cytokine treatment and highlight the importance of understanding the effects of cytokines on melanoma to undergo MT.