Mass Spectrometry: Research to Clinical Diagnostics Agenda

Automated, Multiplexed Quantitation of Proteins; Facilitating Mass Spectrometric Assays in Research and in the Clinic
Selena Larkin, Vice President, Marketing and Sales, SISCAPA/Omicia, United States of America

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.

Bioanalytical Sample Preparation Strategies Prior to LC-MS/MS Analysis of Drugs and Biomarkers
Vincenzo Pucci, Principal Scientist, Preclinical Bioanalytical Group, Merck Research Laboratories, United States of America

An essential component of the drug-discovery process is the determination of drug and biomarker concentrations in biological samples which yields the data necessary to understand the pharmacokinetics and pharmacodynamic, respectively. To obtain these data, each sample of fluid obtained from an in vitro or in vivo study is subjected to sample preparation, chromatographic separation, and analytical detection. Sample preparation is an important and necessary step in the overall analytical process, because most analytical instruments cannot accept the sample matrix directly. Despite the tremendous advances in liquid chromatography mass spectrometry (LC-MS) and LC tandem mass spectrometry (LC-MS/MS) detection systems in recent times, sample preparation still represents the most challenging part of the bioanalytical workflow. The removal of unwanted endogenous matrix components (e.g., protein, salts, etc.) from the sample must be rapid and as complete as possible to avoid the introduction of interferences upon analysis. Many different techniques are available to a drug discovery scientist performing bioanalytical sample preparation. The focus of this presentation is to introduce the goals for sample preparation and the many choices available to the drug-discovery scientist.