Automated, Multiplexed Quantitation of Proteins; Facilitating Mass Spectrometric Assays in Research and in the ClinicWednesday, 30 September 2015 at 16:30 Add to Calendar ▼2015-09-30 16:30:002015-09-30 17:30:00Europe/LondonAutomated, Multiplexed Quantitation of Proteins; Facilitating Mass Spectrometric Assays in Research and in the ClinicNGS, SCA, Mass Spec: The Road to Diagnostics in San Diego, California, USASan Diego, California, USASELECTBIOenquiries@selectbiosciences.com Proteomics workflows often lack the necessary reproducibility required for clinical applications. This is in part due to the complex nature of these workflows, which typically start with enzymatic digestion (e.g. trypsin) of complex specimens (e.g. human blood) followed by multiple fractionation steps prior to analysis by a mass spectrometer. Therefore, in order to comply with the clinical specifications for total analytical error, a simple and reproducible workflow is needed. Here we report the development of a generic, highly reproducible tryptic digestion method for complex protein mixtures. This digestion approach is further complemented with the use of the SISCAPA technology (sequence-specific capture of target peptides and corresponding stable isotope labeled standards using high-affinity anti-peptide antibodies from digested specimens). The result is an ‘addition-only’ or homogeneous approach that can be easily implemented on a variety of automated liquid handling platforms for precise, robust and sensitive measurement of peptide analytes. The addition-only strategy allows for sequential addition of samples and reagents in a 96-well format and eliminates the need for separative techniques such as filtration, solid phase extraction or centrifugation. Furthermore, the SISCAPA enrichment of analytes creates eluted samples that can be analyzed by mass spectrometry with minimal liquid chromatography. We have implemented this workflow on a Bravo Liquid Handling Robot (Agilent Technologies, CA) for liquid specimens (e.g. whole blood, plasma/serum) and dried blood spots (DBS). In collaboration with Agilent Technologies, we have also designed an intuitive user interface that further simplifies the workflow from an operational perspective. |