title: Comparison of one- and two-dimensional liquid chromatography approaches in the label-free quantitative analysis of Methylocella silvestris.
abstract: The proteome of the bacterium Methylocella silvestris has been characterized using reversed phase ultra high pressure liquid chromatography (UPLC) and two-dimensional reversed phase (high pH)-reversed phase (low pH) UPLC prior to mass spectrometric analysis. Variations in protein expression levels were identified with the aid of label-free quantification in a study of soluble protein extracts from the organism grown using methane, succinate, or propane as a substrate. The number of first dimensional fractionation steps has been varied for 2D analyses, and the impact on data throughput and quality has been demonstrated. Comparisons have been made regarding required experimental considerations including total loading of biological samples required, instrument time, and resulting data file sizes. The data obtained have been evaluated with respect to number of protein identifications, confidence of assignments, sequence coverage, relative levels of proteins, and dynamic range. Good qualitative and quantitative agreement was observed between the different approaches, and the potential benefits and limitations of the reversed phase-reversed phase UPLC technique in label-free analysis are discussed. A preliminary screen of the protein regulation data has also been performed, providing evidence for a possible propane assimilation route.
authors.affiliation: School of Life Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom.
authors.forename: Nisha A
authors.initials: NA
authors.lastname: Patel
authors.forename: Andrew
authors.initials: A
authors.lastname: Crombie
authors.forename: Susan E
authors.initials: SE
authors.lastname: Slade
authors.forename: Konstantinos
authors.initials: K
authors.lastname: Thalassinos
authors.forename: Chris
authors.initials: C
authors.lastname: Hughes
authors.forename: Joanne B
authors.initials: JB
authors.lastname: Connolly
authors.forename: James
authors.initials: J
authors.lastname: Langridge
authors.forename: J Colin
authors.initials: JC
authors.lastname: Murrell
authors.forename: James H
authors.initials: JH
authors.lastname: Scrivens
mesh_terms: D001426:Bacterial Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D042062:Beijerinckiaceae / Q000737:chemistry* / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D003470:Culture Media; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D008697:Methane / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D011407:Propane / Q000378:metabolism; D019802:Succinic Acid / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D001426:Bacterial Proteins; D003470:Culture Media; D010446:Peptide Fragments; D019802:Succinic Acid; D008697:Methane; D011407:Propane