Over the past decade, a suite of new mass-spectrometry-based proteomics methods has been developed that now enables the conformational properties of proteins and protein-ligand complexes to be studied in complex biological mixtures, from cell lysates to intact cells. Highlighted here are seven of the techniques in this new toolbox. These techniques include chemical cross-linking (XL-MS), hydroxyl radical footprinting (HRF), Drug Affinity Responsive Target Stability (DARTS), Limited Proteolysis (LiP), Pulse Proteolysis (PP), Stability of Proteins from Rates of Oxidation (SPROX), and Thermal Proteome Profiling (TPP). The above techniques all rely on conventional bottom-up proteomics strategies for peptide sequencing and protein identification. However, they have required the development of unconventional proteomic data analysis strategies. Discussed here are the current technical challenges associated with these different data analysis strategies as well as the relative analytical capabilities of the different techniques. The new biophysical capabilities that the above techniques bring to bear on proteomic research are also highlighted in the context of several different application areas in which these techniques have been used, including the study of protein ligand binding interactions (e.g., protein target discovery studies and protein interaction network analyses) and the characterization of biological states.

Capsule Summary RNA-seq and denaturation studies demonstrated that allergens are among the most highly expressed and stable proteins from Dermatophagoides pteronyssinus. The combination of high levels of transcription and stability for the major allergens Der p 1, Der p 2, and Der p 23 is rare.

The proteins in an MCF-7 cell line were probed for tamoxifen (TAM) and n-desmethyl tamoxifen (NDT) induced stability changes using the Stability of Proteins from Rates of Oxidation (SPROX) technique in combination with two different quantitative proteomics strategies, including one based on SILAC and one based on isobaric mass tags. Over 1000 proteins were assayed for TAM- and NDT- induced protein stability changes, and a total of 163 and 200 protein hits were identified in the TAM and NDT studies, respectively. A subset of 27 high confidence protein hits were reproducibly identified with both proteomics strategies and/or with multiple peptide probes. One-third of the high confidence hits have previously established experimental links to the estrogen receptor, and nearly all of the high confidence hits have established links to breast cancer. One high confidence protein hit that has known estrogen receptor binding properties, Y-box binding protein 1 (YBX1), was further validated as a direct binding target of TAM using both the SPROX and pulse proteolysis techniques. Proteins with TAM- and/or NDT-induced expression level changes were also identified in the SILAC-SPROX experiments. These proteins with expression level changes included only a small fraction of those with TAM- and/or NDT-induced stability changes.

To the Editor, The human exposome contains millions of proteins, yet only a select few consistently become allergens. This has led to speculation that there are intrinsic factors which differentiate allergens from non-allergens. Anecdotal evidence suggested that abundance and stability could be two such properties, but no rigorous statistical comparisons were available until a study of the dust mite Dermatophagoides pteronyssinus (Dp) combined transcriptomics with proteomics. 1 The Dp study measured transcription levels as a proxy for protein abundance and correlated the results with proteome-wide stability measurements using mass spectrometry. It was found that the mite allergens were as a population more stable and more highly expressed than Dp non-allergens. Dp was a good model

In medicinal chemistry, one of the most studied molecules in recent history is taxol. Taxol is a versatile natural product that is used in various cancer treatment regimens. It is administered to patients with breast, lung, and ovarian cancers, and is currently being studied for the treatment of squamous cell carcinoma of the oral cavity and tongue. Taxol has been tested in a number of research and clinical phase trials to determine feasibility, toxicity, and cytotoxicity against oral squamous cell carcinoma as a single drug regimen and as a contributing drug component in treatment plans. This paper reviews over forty articles that examine cell lines, murine models, and human results for the response of taxol against squamous cell carcinoma (SCC) of the oral cavity and the tongue.