Assuntos
Alérgenos , Hipersensibilidade , Poeira , Humanos , Hipersensibilidade/epidemiologia , Hipersensibilidade/etiologia , PólenRESUMO
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.
Assuntos
Espectrometria de Massas/métodos , Processamento de Proteína Pós-Traducional , Proteínas/química , Proteoma/química , Proteômica/tendências , Animais , Reagentes de Ligações Cruzadas/química , Bases de Dados de Proteínas , Medição da Troca de Deutério/métodos , Humanos , Marcação por Isótopo/métodos , Ligantes , Espectrometria de Massas/instrumentação , Ligação Proteica , Dobramento de Proteína , Estabilidade Proteica , Proteínas/metabolismo , Proteínas/ultraestrutura , Proteólise , Proteoma/ultraestrutura , Proteômica/instrumentação , Proteômica/métodos , Análise de Sequência de Proteína/instrumentação , Análise de Sequência de Proteína/métodos , Análise de Sequência de Proteína/estatística & dados numéricos , TermodinâmicaRESUMO
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.