Proteomes dynamic range

The enormous dynamic range of proteins in the sample represents an additional difficulty in proteome analysis. The best example is semm with a protein abundance ranging over eleven orders of magnitude (Anderson and Anderson, 2002). To detect the low abundant species, one has to load a sufficient amount of digest on a column to meet the limit of detection (LOD) of the MS instrument. Some reports published used up to 2.5 L of plasma with an extensive fractionation of intact proteins prior to LC-MS analysis on the peptide level (Rose et al., 2004). 

Figure 7.6. (a) Definitions of success rate and relative dynamic range, (b) Model of a proteomics experiment. (See color insert.)... 

The dynamic range of protein expression represents a main obstacle since abundant proteins are seldom of interest and others such as transcription factors are only present in a few copies. There is no detector that is able to visualize all proteins at the same time so that prefractionation and the investigation of subproteomes is required. In fact, pre-MS sample preparation techniques exploiting electrophoretic, chromatographic, or chemical properties of the analyte are often the bottleneck of proteomics. 

Upon use of structurally modified variants as internal standards for the particular analytes, the relative quantificahon of oligonucleotides, peptides, and small proteins was demonstrated [44]. The potential of the ILM to allow quantitative analyses of peptides without the use of internal standards was presented recently [43]. Linear correlahons between peptide amount and signal intensities could be found upon applicahon of increased matrix-to-analyte ratios between 25,000 and 250,000 (mokmol). The dynamic range of linearity thus spanned one order of magnitude. Unfortunately, the importance of the M/A ratio prevents the use of this method in samples with unknown orders of concentration, for example, in a proteomics environment. On the other hand, the method is applicable for the screening of enzyme-catalyzed reactions because the starting concentrahons of the peptides are generally known in such assays. 

One clear aspect of the current trends in the proteomic field is the plethora of ways to achieve informational outcomes. Research developments over recent years have focused on all facets of the proteomic workflow including improvements in the comprehensiveness of analysis, efficiency of workflows, and development of specialized methods. This is aided by several mass spectrometers being available on the market that have the necessary accuracy, sensitivity, speed, and dynamic ranges. The baseline workflow for proteomics however remains essentially the same and involves a number of required steps (summarized in the succeeding text and in Figure 1) ... 

Corthals GL, Wasinger VC, Hochstrasser DF et al (2000) The dynamic range of protein expression a challenge for proteomic research. Electrophoresis 21 1104—1115... 

Table 3.1 The most commonly used protein staining methods for proteome analysis using 2-DE. Besides the low detection limit of the applied staining methods, the linear dynamic range for protein quantification is another important parameter for a global description of a proteome.

Multidimensional liquid chromatography (MD-LC) has not yet developed as a front-end technique in proteomics due to the lack of fundamental understanding on how to handle complex sample mixtures with an enormously wide abundance and dynamic range. 

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