title: Ultra-fast tandem mass spectrometry scanning combined with monolithic column liquid chromatography increases throughput in proteomic analysis.
abstract: Liquid chromatography combined with electrospray ionization mass spectrometry (LC/ESI-MS) has been used successfully for the characterization of biomolecules 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 one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (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 post-translational modifications (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.
authors.affiliation: Henry Wellcome Building for Molecular Physiology, Nuffield Department of Clinical Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK.
authors.forename: Mariola
authors.initials: M
authors.lastname: Batycka
authors.forename: Neil F
authors.initials: NF
authors.lastname: Inglis
authors.forename: Ken
authors.initials: K
authors.lastname: Cook
authors.forename: Alex
authors.initials: A
authors.lastname: Adam
authors.forename: Douglas
authors.initials: D
authors.lastname: Fraser-Pitt
authors.forename: David G E
authors.initials: DG
authors.lastname: Smith
authors.forename: Laura
authors.initials: L
authors.lastname: Main
authors.forename: Anneke
authors.initials: A
authors.lastname: Lubben
authors.forename: Benedikt M
authors.initials: BM
authors.lastname: Kessler
mesh_terms: D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation* / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism; D008832:Microchemistry / Q000295:instrumentation / Q000379:methods; D010449:Peptide Mapping; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*; D012680:Sensitivity and Specificity; D012709:Serum Albumin / Q000032:analysis; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013997:Time Factors
chemical_list: D012709:Serum Albumin