In the search for viable drug targets and compounds, drug discovery researchers are adopting high-resolution, high-content protein identification and functional analysis methods. Liquid chromatography combined with mass spectrometry (LC-MS) offers the resolving power and structural information about proteins needed to address these applications and others in phosphopeptide analysis, biomarker discovery, and metabolomics.
For example, mass spectral comparison of protein mixtures from diseased versus healthy tissue samples can provide deep insight into the molecular nature of diseases and uncover biomarkers and biomarker patterns of therapeutic and diagnostic value. Sensitive, high-resolution LC-MS can lead directly to the identification of drug target proteins, and enable researchers to detect and quantify therapeutic and toxic effects of potential new chemical entities.
Efforts to optimize LC-MS technology to meet the escalating requirements of proteomics researchers for analytical speed, sensitivity, and reproducibility have led to the development of nanoscale proteomics and nanoflow chromatography, a method that takes advantage of small sample quantities and low chromatographic solvent flow rates.
Low-volume, low-flow-rate chromatography improves peptide identification and simplifies mass spectrometry analysis by delivering separated components at high peak concentrations to a nano electrospray ionization source. This enhances the sensitivity of mass spectrometry, enabling the analysis of smaller samples of purified protein preparations. When implemented with components specifically matched to the requirements of nanoflow chromatography, nanoscale HPLC separation systems also reduce solvent waste by orders of magnitude.
The main challenge of nanoflow chromatography is maintaining flow stability at low flow rates. Optimal flow rates for nanoscale LC columns are between 100 and 500 nanoliters per minute. Conventional HPLC pumps are optimized for flow rates of three to four orders of magnitude higher than this, and instrument manufacturers initially entered the market for nanoflow chromatography with systems based on the use of these pumps combined with flow splitters, which bleed off all but a small amount of the flow.
For routine separations at standard HPLC flow rates, conventional, piston-driven HPLC pumps provide accurate flow rates, but can suffer from pulsing due to piston restroke and typically require additional hardware to dampen this effect. The flow rate stability of systems based on flow-splitting delivery formats is further degraded by backpressure changes, which can cause the actual flow rate to the column to deviate substantially from the set point.
The cumulative effect of these limitations is reduced separation reproducibility and an inability to perform precise gradients at the low flow rates needed to optimize the sensitivity advantages of nanoscale chromatography systems.