With the recent availability and accessibility of mass spectrometry for basic and clinical research, the requirement for stable, sensitive, and reproducible assays to specifically detect proteins of interest has increased. Multiple reaction monitoring (MRM) or selective reaction monitoring (SRM) is a highly selective, sensitive, and robust assay to monitor the presence and amount of biomolecules. Until recently, MRM was typically used for the detection of drugs and other biomolecules from body fluids. With increased focus on biomarkers and systems biology approaches, researchers in the proteomics field have taken advantage of this approach. In this chapter, we will introduce the reader to the basic principle of designing and optimizing an MRM workflow. We provide examples of MRM workflows for standard proteomic samples and provide suggestions for the reader who is interested in using MRM for quantification.
Background: Epitope mapping is an increasingly important aspect of
biotherapeutic and vaccine development. Recent advances in therapeutic
antibody design and production has enabled candidate mAbs to be
identified at a rapidly increasing rate resulting in a significant
bottleneck in the characterization of ‘structural’ epitopes, that are
challenging to determine using existing high throughput epitope mapping
tools. Here, Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS)
epitope screening workflow was introduced that is well suited for
accelerated characterization of epitopes with a common antigen. Main
methods and major results: The method is demonstrated on set of 6
candidate mAbs targeting Pertactin (PRN). Using this approach, five of
the six epitopes was unambiguously determined using two HDX mixing
timepoints in 24 hours total run time, corresponding to substantial
decrease in the instrument time required to map a single epitope using
conventional HDX workflows. Conclusion: An accelerated HDX-MS epitope
screening workflow was developed. The two-timepoint ‘screening’ workflow
mapped all six mAbs and generated high confidence epitopes for five of
the six mAbs assayed. The substantial improvement in the rate of data
collection can advance HDX-MS for higher throughput investigations
supporting the ability to evaluate a broader number of mAb candidates at
an earlier stage of vaccine development.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.