Advanced MudPIT as a next step toward high proteome coverage

We present a simple, time‐ and cost‐efficient approach to tackle the proteome of prokaryotic organisms. To obtain large data sets of complex biological experiments high‐throughput and time‐ and cost‐efficient methods still have to be developed and refined. In this study, we combined well‐approved te...

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Bibliographic Details
Published in:Proteomics (Weinheim) 2011-09, Vol.11 (18), p.3651-3656
Main Authors: Fränzel, Benjamin, Wolters, Dirk A.
Format: Article
Language:English
Subjects:
Algorithms
Analytical, structural and metabolic biochemistry
Bacterial Proteins - analysis
Bacterial Proteins - chemistry
Bacteriology
Biological and medical sciences
Cell Membrane - chemistry
Chromatography, High Pressure Liquid - instrumentation
Chromatography, High Pressure Liquid - methods
Competitiveness
Corynebacterium glutamicum
Corynebacterium glutamicum - chemistry
Corynebacterium glutamicum - growth & development
Cytosol - chemistry
Data acquisition
Data processing
Elevated temperature
Escherichia coli
Escherichia coli - chemistry
Escherichia coli - growth & development
Fundamental and applied biological sciences. Psychology
Gram-negative bacteria
Improved MudPIT
Mass Spectrometry - instrumentation
Mass Spectrometry - methods
Membrane proteins
Membrane Proteins - analysis
Membrane Proteins - chemistry
Membrane proteome coverage
Miscellaneous
Proteins
Proteome - analysis
Proteome - chemistry
proteomics
Software
Subcellular Fractions - chemistry
Technology
Temperature
Temperature effects
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Summary:We present a simple, time‐ and cost‐efficient approach to tackle the proteome of prokaryotic organisms. To obtain large data sets of complex biological experiments high‐throughput and time‐ and cost‐efficient methods still have to be developed and refined. In this study, we combined well‐approved techniques, namely elevated chromatographic temperatures, long RP columns and the multidimensional protein identification technology MudPIT to achieve high proteome coverage. The advanced MudPIT approach has been evaluated and delivered very comprehensive results for Gram‐positive as well as Gram‐negative bacteria (53% proteome coverage for Corynebacterium glutamicum and 46% proteome coverage for Escherichia coli). Also, a high identification rate for the challenging integral membrane proteins was achieved. The competitiveness of the advanced MudPIT technology is strengthened by the fact that in this approach only two fractions were analyzed with both, simple and time‐efficient sample preparation, and a moderate data acquisition time.
ISSN:1615-9853
1615-9861
DOI:10.1002/pmic.201100056