ProteinPilot software

The IκB kinase (IKK) pathway is important in tumorigenesis and is known to be misregulated in a number of cancers including breast cancers. IKK is primarily known for its central role in the activation of NF-κB, which may contribute to tumourigenesis through regulation of the cell cycle and evasion of apoptotic signals. In this research study, the effect of a specific IKK inhibitor, IMD0354, on the T47D breast cancer cell line was investigated. T47D cells are an ER+ breast cancer cell line with an intact IKK signaling pathway.

On-line two-dimensional (2D) liquid chromatography is widely used for protein identification and quantification because of the advantage of increased peak capacity. A common workflow is the combination of strong cation exchange (SCX) as an orthogonal first dimension to reversed-phase (RP) chromatography as the second dimension. A more recently developed strategy has emerged where the first dimension is a high pH RP separation which has less orthogonality, but provides higher peak capacity compared to SCX. These 2D workflows are essential for measuring lower abundant proteins in complex proteomes, however can be complex to implement. In this application note, a simplified chip based 2D-LCMS workflow using a high pH/RP first dimensional separation and a low pH/RP secondary dimension coupled directly to mass spectrometer for proteomic analysis is described.

NanoLC/MS/MS is the current method of choice for high sensitivity identification of proteins, e.g. for the discovery of biomarkers in plasma or other biological fluids. The solvent system typically used is determined by the requirements for both the chromatography and the electrospray processes. While it has been demonstrated that solvents like DMSO can increase electrospray ionization efficiency and improve sensitivity, the potential adverse effect on chromatographic separation has prevented researchers from adding these to the mobile phases. Postcolumn addition of these solvents is a good alternative, but not easy to achieve in nanoLC because of the unavoidable introduction of dispersion using a post-column Tee and additional connections. This poster introduces an extension to a chip-based nanoLC separation device that includes a Tee that is directly integrated into the chip post-column.

Monoclonal antibodies (mAb) are major target-oriented biotherapeutics used to treat an array of human diseases. This poster explores the application of an unbiased and deterministic, data-independent method for mAb analysis using SWATH™. This approach provides substantial benefits over other MS strategies because it captures comprehensive, quantitative MS and MS/MS information for every analyte in the sample, in every analytical run.

In recent years MS has emerged as a superior method for characterizing the heterogeneity of monoclonal antibodies. This application note presents a high-speed LC/MS/MS method for the analysis of enzymatically digested therapeutic antibodies. Sophisticated software tools, that enable more accurate peptide mapping, are also described.