P202-S Expanding the Capabilities of Peptide MRM-Based Assays in Plasma Using a Hybrid Triple-Quadrupole Linear Ion-Trap Mass Spectrometer

As the study of protein biomarkers increases in importance, technical limitations to the detection of low-abundance proteins and high-throughput, high-precision quantitation remain to be overcome. The complexity and dynamic range of the plasma proteome makes the task of specific, quantitative detection even more challenging. Multiple reaction monitoring (MRM) capabilities of triple quadrupole MS systems have been explored as solutions to this challenge due to their well-known sensitivity and selectivity for components in complex matrices such as plasma. Recently, a suite of >100 MRMs representing ~50 plasma protein markers were monitored quantitatively in a single assay using the MRM-based technique showing detection of proteins down to the level of L-selectin (~1μg/mL) with minimal sample preparation and no peptide or protein standards for most of the plasma protein markers.¹As more extensive candidate biomarker panels are being identified, MRM assays will need to be more rapidly developed to verify the expression changes of these proteins across larger clinical sample sets. To do this, the unique combination of triple-quadrupole and ion-trapping capabilities of the hybrid triple quadrupole–linear ion trap mass spectrometer have been utilized. A strategy for rapid MRM assay development for larger-scale profiling and qualification of biomarker candidates without having to first prepare synthetic peptide standards is currently being investigated and involves a chemical labeling strategy to create global reference standards to enable quantitative comparisons between clinical samples. Single assays consisting of ~500s of MRM transitions have been developed for this rapid qualification phase, facilitated by intelligent use of retention time windows during an LC analysis, while maintaining an optimum number of data points for improved precision of peak area and quantitative profiling. This presentation will demonstrate the details of this workflow with human plasma examples.