Abstract
Proteomics became a key tool for the study of biological systems. The comparison between two different physiological states allows unravelling the cellular and molecular mechanisms involved in a biological process. Proteomics can confirm the presence of proteins suggested by their mRNA content and provides a direct measure of the quantity present in a cell. Global and targeted proteomics strategies can be applied. Targeted proteomics strategies limit the number of features that will be monitored and then optimise the methods to obtain the highest sensitivity and throughput for a huge amount of samples. The advantage of global proteomics strategies is that no hypothesis is required, other than a measurable difference in one or more protein species between the samples. Global proteomics methods attempt to separate quantify and identify all the proteins from a given sample. This review highlights only the different techniques of separation and quantification of proteins and peptides, in view of a comparative and quantitative global proteomics analysis. The in-gel and off-gel quantification of proteins will be discussed as well as the corresponding mass spectrometry technology. The overview is focused on the widespread techniques while keeping in mind that each approach is modular and often recovers the other.
Highlights
The ability of detecting significant differences between two cellular states is a universal approach to unravelling the cellular and molecular mechanisms involved in a process with an ultimate goal of discovering new markers, diagnostics and indirectly to track new therapeutic routes
In order to reduce the complexity of the samples, the proteins can be separated by electrophoresis, chromatography, proteome fractionation based on differential solubility, and by aqueous two-phase system (ATPS)
In the spectral counting label-free quantification method [76], the relative quantification of proteins is based on the comparison of the number of MS or tandem MS (MS/MS) spectra performed for a given protein between several experiments
Summary
The ability of detecting significant differences between two cellular states is a universal approach to unravelling the cellular and molecular mechanisms involved in a process with an ultimate goal of discovering new markers, diagnostics and indirectly to track new therapeutic routes. The second step corresponds to the sample preparation (extraction, concentration, purification to remove contaminants such as lipids or nucleic acids, and storage of proteins) while the third is related to methods of separation, and the fourth to quantification and identification of proteins [8] (Figure 1). The top-down strategy is based on the MS analysis of entire proteins [11] The latter is a targeted approach allowing the identification of proteins but especially more comfortably characterisation of isoforms, post translational modifications (PTMs) or conducting of structural studies. It needs significant amounts of biological samples as well as the separation and isolation of intact proteins. The reader can refer to a recent book which gives a detailed survey of the quantitative methods in proteomics [12]
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