High Throughput Quantitative Mass Spectrometry

Since the advent of atmospheric pressure ionization sources, liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) has become the indisputable gold standard technique for quantitative analysis for in vitro and in vivo biological samples. Dramatic improvements in throughput by LC-MS/MS have been achieved based on need and confluences of technologies over the past 20 years primarily in mass spectrometry (MS) and liquid chromatography (LC) augmented with improvements in automation and robotics. Evolutions in mass spectrometer sampling interfaces, mass analyzers, ion optics, and electronics have enabled the development of a rugged, reliable, and exquisitely sensitive platform. Instrument-to-instrument variation now is controlled such that instrument parameters can be translated in a facile manner to like platforms from the same manufacturer. Continued increases in MS instrument sensitivity have enabled reductions in sample loading needs, a key requirement for robust, hands-free operation. Instrument tuning algorithms and sample delivery workflows have been developed to automatically create acquisition methods for chosen analytes to enable streamlining of method development and sample analysis. Systematic evolutions in LC pumps and column hardware have led to better sensitivity, improved chromatographic resolution with a concomitant reduction in analysis time, and robustness. Numerous techniques that rely on robust chromatography have been developed to increase the effective data collection rate of mass spectrometers, the time spent acquiring data of value. In a single decade, we have seen evolution from the serial delivery mode of a single LC system coupled to a mass spectrometer up to commercial versions of eight-channel “true” parallel and eight-channel serial (staggered) parallel LC systems coupled to a single mass spectrometer. Before sample analysis, generic automated sample preparation techniques have been developed. Robotics capabilities have improved sample handling processes for protein and phospholipid removal along with sample pooling, through the use of 96- and 384-well-plate-based systems. Postacquisition data processing and reporting solutions have also been developed to handle the greater influx of data generated by this high throughput platform. When these developments are taken into account, we are left with the realization that quantitative MS has become an industrialized process. We now have dramatically shortened the timelines required for analytical support to the point it is not a rate-limiting step in the discovery process. This chapter discusses the evolution of high throughput LC-MS/MS and many of the approaches that have been pursued in the past decade. We also consider non-LC-based workflows that utilize alternative ionization sources. With some of these new strategies, it is conceivable that data acquisition rates will reach or exceed multiple assays per second. Keywords: liquid chromatography-mass spectrometry (LC-MS); tandem mass spectrometry (MS/MS); high throughput; triple quadrupole; electrospray; atmospheric pressure ionization; selective reaction monitoring; automation