Tempo™ ht LC System
3Pages

Christie L. Hunter > 1 1 , and David M. Cox > 2 2 Applied Biosystems, USA, MDS Sciex,Canada From simple protein identification experiments to global protein expression workflows, proteomic applications require sensitive, reproducible, reliable chromatography. Tempo nano LC systems set a new performance standard for low flow separations. > The Tempo nano LC systems employ a direct pumping system with flow meters monitoring the flow rate in each mobile phase to a precision of <1 nL/min. Flow rate information is continuously fed to a microfluidic flow controller which rapidly adjusts a variable pressure source. This allows the system to maintain precise flow rates from 20-1000 nL/min. Each mobile phase is monitored independently before mixing, providing for precise gradient formation (Figure 1). The simplicity in design of the Tempo nano LC systems represents a significant improvement in ease-of-use and reliability for the proteomics researcher. This direct pumping system eliminates the need for flow splitting, alleviating the flow inaccuracies of splitter based systems and reducing solvent consumption and waste. Close coupling of the pump, gradient mixer and autosampler minimizes dispersion and delay time. Flow rate is unaffected by downstream pressure fluctuations, increasing the long term run to run stability of these low flow separations. > Օ Direct pumping system and the measurement of flows with active feedback ensures precise gradient delivery, providing excellent run-to-run reproducibility Optimized for low flow rates between 201000 nL/minute, (and 1-20 ֵ L/min for the first dimension pump in MDLC system) Օ Ultra low delay volume of 65 nL from mixer to injection valve enables rapid gradients and re-equilibration to shorten total cycle times Elimination of flow splitting reduces operating costs due to low solvent consumption and minimal waste production Full MDLC functionality available Օ Easy-to-use software interface Simplified plumbing scheme and split-less design make for a reproducible, easy-to-use system > Figure 1. Real-time monitoring of flow ratesfor each mobile phase at 500 nL/min. Microfluidic flow control provides a high level of flow accuracy for each channel, enabling very precise gradient formation. www.appliedbiosystems.com >

Many LCMS applications, such as biomarker discovery by MS profiling, metabolomics, peptide quantitation, etc., require the comparison of chromatographic peaks across many replicate injections. Quantitative peptide/protein analysis in complex mixtures is typically done using multiple reaction monitoring (MRM) on a 4000 Q TRAP m C18 column (PepMap C18, 300 nL/min), peak widths at half height of 6-10 seconds were observed with excellent peak shape (Figure 2). The retention time of each peak was measured and the variation was calculated for each peak. An average standard deviation of 0.08...

The analysis of many complex samples in a highly reproducible manner is a key component of protein biomarker discovery using MS profiling as well as for the validation of protein biomarkers. The reproducibility of an LC MRM method measuring 38 proteins in a single run was assessed by measuring 10 LC-MRM replicates of the same sample of either digested or depleted digested human plasma on a 4000 Q TRAP > system. The equivalent of 10 nL of plasma (digested with trypsin) was loaded directly onto a 75 ε m C18 column (PepMap, 300 nL/min) and eluted using a rapid 30 minute gradient. With no...