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 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.