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J Biomol Tech. 2010 September; 21(3 Suppl): S31.
PMCID: PMC2918048

High Throughput and Precision for Nano-Liquid Chromatography Using an Automated Heated Dual Column Nanospray Source

A. Fogiel, K. Heaton, A. Fogiel, Jr., L. Heineman, and S.L. Staats
Phoenix S&T, Chester, PA, United States



Nano LC-MS is the technique of choice for clinical proteomics because of its high sensitivity and data content.Common experimental aberrations in nanospray-MS such as loss of spray, sputtering, and emitter degradation, etc. during gradient-LC severely impact the technique's capability to obtain reliable and reproducible results from precious clinical samples. Constant monitoring of and manual intervention during a nano LC-MS run have become standard practice.Moreover the time required to completely flush out the sample in the biological matrix and the long column equilibration time make throughput undesirably low.We present an automated nano LC-MS source that enables reliable, reproducible and completely unattended operation at doubled the throughput.The source accommodates two nano LC columns: while a gradient LC-MS experiment is performed in column A, column B is equilibrating and after equilibration, loading sample off axis from the mass spectrometer inlet to prevent the dirty matrix from contaminating the MS.Before each column begins the LC run, the spray emitter undergoes a short series of spray emitter conditioning which includes wiping and a nitrogen purge to ensure an optimized spray during the experiment.The results presented are on the dual-column separation of a 63-peptide mixture with and without a plasma matrix.The flow rate was 270nL/min provided by a gradient pump for the LC and an isocratic pump for the sample loading and column equilibration.The precision and reproducibility of the mass chromatograms were further enhanced by heating the columns at 50C.Retention time precision with average RSD of 0.5% was achieved over three runs on both columns on neat samples.The column heating also contributed to better peak separations.The potential of further increasing throughput will be discussed.We thank Bryan Krastins and David Sarracino of ThermoFisher for providing the samples and for valuable discussions.

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