Comparison of One- and Two-dimensional Liquid Chromatography Approaches in the Label-free Quantitative Analysis of Methylocella silvestris
| Autor: | Joanne B. Connolly, Nisha A. Patel, Susan E. Slade, James I. Langridge, Andrew T. Crombie, James H. Scrivens, Chris Hughes, Konstantinos Thalassinos, J. Colin Murrell |
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| Rok vydání: | 2012 |
| Předmět: |
Succinic Acid
Analytical chemistry Fractionation Tandem mass spectrometry Biochemistry High-performance liquid chromatography Propane Bacterial Proteins Beijerinckiaceae Tandem Mass Spectrometry Electrophoresis Gel Two-Dimensional Chromatography High Pressure Liquid Chromatography Reverse-Phase Chromatography biology Methylocella silvestris Chemistry Substrate (chemistry) General Chemistry biology.organism_classification Peptide Fragments Culture Media Electrophoresis Proteome Methane Quantitative analysis (chemistry) |
| Zdroj: | Journal of Proteome Research. 11:4755-4763 |
| ISSN: | 1535-3907 1535-3893 |
| DOI: | 10.1021/pr300253s |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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