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J Biomol Tech. 2010 September; 21(3 Suppl): S58.
PMCID: PMC2918042

Monolithic Columns for High-efficiency LC-MS/MS Peptide Mapping

G. Gendeh2 and S. Eeltink1
1Dionex Corporation, Amsterdam, The Netherlands;
2Dionex Corporation, Sunnyvale, CA, United States



Peptide MS analysis applied in proteomics research has made great advances over the past years in terms of sensitivity and scan speeds. However, in order to truly tackle present-day proteomic samples, different strategies have to be followed to increase the separation performance, including the development of novel column technologies. Polymer monolithic columns have become an attractive alternative for packed columns. Especially for the nanoLC-MS analyses of complex biological samples they offer high-efficiency separations due to the absence of inter-particle mass transfer. The porosity of monolithic materials can be influenced to tune the permeability and reaching maximum separation efficiency. Optimization of the monolithic structure allows preparation of very long columns (1 meter), while maintaining the column pressure at an acceptable level. The performance of nanoLC columns upto 1 m in length was tested by the gradient-elution of complex tryptic digests. Experiments were conducted at low and high pH mobile phases with different ion-pairing agents. MS detection was conducted by electrospray interfacing with an ion-trap mass spectrometer operated in positive and negative ion mode. The peak capacity was experimentally determined by averaging the peak width at 4? for at least ten peptides. The maximum peak capacity on long poly(styrene-co-divinyl benzene) monolithic nanoLC columns was obtained at much longer (shallower) gradients than typically applied for conventional (5 cm long) monolithic columns. Peak capacities exceeding 1000 were achieved when using 1 m long monolithic columns with optimized morphology and applying 1 -5 h gradients.

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