g by SDS-PAGE [7] or chromatography,

and depletion of ab

g. by SDS-PAGE [7] or chromatography,

and depletion of abundant proteins [8, 9]. The accurate quantitation of changes in protein expression in or between different samples Selleckchem EGFR inhibitor or states is one of the primary objectives in proteomics [10]. Several methods for labeling proteins metabolically (in cell cultures) or after extraction are widely applied in “”shotgun”" proteomics. The labels either incorporate heavy, stable isotopes or a fluorescent group. Nonetheless, it is also possible to quantify peptides and proteins in individual samples directly from the mass spectrometer signal, the so-called “”label-free”" quantitation. This type of quantitation demands reproducible sample preparation and protein digestion, and benefits from using a mass spectrometer with a wide dynamic range and resolving power, such as an FTICR instrument. Despite these prerequisites, label-free quantitation holds a few advantages over the use of labels. For instance, the sample workup procedure is simpler as there is no

labeling step, and the number of samples is not in any way limited by number of labeling reagents and can be used in large studies or for analyzing a large number of time points. Methods based on labeling, on the other Selleck GSK2126458 hand, have a built-in maximum number of samples that can be analyzed in parallel, beyond which multiple analyses has to be made by bridging between them (which requires one sample or reference to be shared between at least two analyses). Label-free methods seek to reduce potential interferences, for instance by increasing resolving power, and improving accuracy, e.g. through data INK128 normalization [11]. In our study we used a novel FTICR-ion trap cluster which combines the high

mass accuracy of FTICR with fast and relatively inexpensive ion traps for MS/MS [12] making it ideally suited for large-scale, label-free proteomic studies. Results and Discussion The from glucose-lactose diauxie is a classical Escherichia coli experiment which has been repeated many times, including recent studies on gene expression using microarrays [13]. In our experimental setup, the growth rate and glucose concentration allowed precise determination of onset of glucose-lactose (Figure 1). The onset of diauxie occurred when cell suspension reached OD600 of ~0.6 or a density of approximately 5 × 108 cells/mL [14]. This was reproducible in each experiment (OD600 of 0.64, 0.60, and 0.55 respectively) and the OD600 could be used as a predictor during the experiment to optimize the sampling of the culture before and during the diauxic shift. The cell density at the onset of diauxic shift was approximately one quarter of the final density, which is consistent with previous observations, and depends on the glucose-lactose ratio [15].

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