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Abstract No.: TuOr-19
Speaker: Benedikt M. Kessler
Session: Peptidomics and Proteomics
Presentation date: Tue, Aug 29, 2006
Presentation time: 16:50 – 17:10

Ultra-Fast MS/MS Scanning Combined with Monolithic Column LC Increases Throughput in Proteomic Analysis

Benedikt M. Kessler1, Mariola Batycka1, Neil F. Inglis2, Ken Cook3, Alex Adam3, Douglas Fraser-Pitt2, David G. E. Smith2, Laura Main4, Anneke Lubben4

1 University of Oxford, Oxford, United Kingdom
2 Moredun Research Institute, Midlothian, United Kingdom
3 Dionex LC Packings, Conventry, United Kingdom
4 Bruker Daltonics, Coventry, United Arab Emirates

Correspondence address: Benedikt M. Kessler, University of Oxford, Clinical Medicine, Roosevelt Dr, Oxford, OX3 7BN United Kingdom.

Web site: http://www.ccmp.ox.ac.uk/kessler/

Keywords: Chromatography, Fast; Chromatography, Liquid -, Nanoscale Capillary; Ionization, Micro Electrospray (Nanospray); MS/MS, Liquid Chromatography.

Novel aspect: Higher throughput capabilities for conventional proteomics applications using monolithic columns combined with ultra-fast MS/MS scanning

 

Liquid chromatography combined with electrospray mass spectrometry (LC-ESI-MS) has been used successfully for the characterization of bio-molecules in proteomics in the last few years. This methodology relied largely on the use of reversed-phase chromatography, in particular C18-based resins, which are suitable for separation of peptides. Here we show that polymeric [polystyrene divinylbenzene] monolithic columns can be used to separate peptide mixtures faster and at a higher resolution. For 500 fmol bovine serum albumin, up to 68% sequence coverage and Mascot Mowse scores of >2000 were obtained using a 9 min gradient on a monolithic column coupled to an ion trap mass spectrometer with ultra-fast MS/MS scan rates. In order to achieve similar results using C18 columns, it was necessary to extend gradient times to 30 min. In addition, we demonstrate the utility of this approach for the analysis of whole Escherichia coli cell lysates by 1D-SDS-PAGE in combination with LC-MS/MS using 4 min gradients on monolithic columns. Our results indicate higher throughput capabilities of monolithic columns (3-fold gain in time or more) for conventional proteomics applications, such as protein identification and high sequence coverage usually required for detection of PTMs. Further optimization of sensitivity and quality of sequence information is discussed, in particular when combined with mass spectrometers that have very fast MS - MS/MS switching and scanning capabilities.