Proteomics Analysis of the Estrogen Effects in the Rat Uterus Using Gel-LC and Tandem Mass Spectrometry Approaches.

Proteomics-based bottoms-up, at a big scale applied to the protein identification and relative quantification present in complex mixtures (cell lysates, tissues, biological fluids, secretome, etc.) is a useful strategy to identify proteins and analyze their changes. Samples processed through a gel-free approach provide a simple method for protein separation and profile comparison of different conditions, such as using fewer steps in the protocol, reducing excessive sample handling, and covering an extended range of molecular weights and isoelectric points. However, it presents a great limitation related to the management of large dynamic ranges of proteins. There are numerous protocols that allow handling the problem or limitations generated by a high dynamic range of the proteins present in the sample. The Gel-LC technique is a complementary alternative of the gel-free approach available to solve the issue of protein samples with a high dynamic range. The different steps of the protocol involve sample processing through Gel-LC (1D-SDS-PAGE) prior to digestion, 1D-nanoUHPLC coupled to high-resolution/mass accuracy tandem mass spectrometry analysis (1D-nanoUHPLC-HR/MA-MS /MS analysis) and afterward, the protein identification and relative quantification analysis using bioinformatics tools for the data conversion, organization, and interpretation.

Proteome Analysis Using Gel-LC-MS/MS.

This article describes processing of protein samples using 1D SDS gels prior to protease digestion for proteomics workflows that subsequently utilize reversed-phase nanocapillary ultra-high-pressure liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS). The resulting LC-MS/MS data are used to identify peptides and thereby infer proteins present in samples ranging from simple mixtures to very complex proteomes. Bottom-up proteome studies usually involve quantitative comparisons across several or many samples. For either situation, 1D SDS gels represent a simple, widely available technique that can be used to either fractionate complex proteomes or rapidly clean up low microgram samples with minimal losses. After gel separation and staining/destaining, appropriate gel slices are excised, and in-gel reduction, alkylation, and protease digestion are performed. Digests are then processed for LC-MS/MS analysis. Protocols are described for either sample fractionation with high-throughput processing of many samples or simple cleanup without fractionation. An optional strategy is to conduct in-solution reduction and alkylation prior to running gels, which is advantageous when a large number of samples will be separated into large numbers of fractions. Optimization of trypsin digestion parameters and comparison to in-solution protease digestion are also described. © 2019 by John Wiley & Sons, Inc.

Proteomic Profiling of Hsp90 Inhibitors.

Mass spectrometry assays demonstrate that Hsp90 inhibitors alter the expression of approximately one-quarter of the assayable proteome in mammalian cells. These changes are extraordinarily robust and reproducible, making "proteomics profiling" the gold standard for validating the effects of new Hsp90 inhibitors on cultured cells. Proteomics assays can also suggest novel hypotheses regarding drug mechanisms. To assist investigators in adopting this approach, this Chapter provides detailed protocols for conducting simple proteomics assays of Hsp90 inhibition. The protocols present a robust label-free approach that utilizes pre-fractionation of protein samples by SDS-PAGE, thereby providing reasonably good penetration into the proteome while addressing common issues with sample quality. The actual programming and operation of liquid chromatography-tandem mass spectrometers is not covered, but expectations for achievable performance are discussed, as are alternative approaches, common challenges, and software for data analysis.