Proteins can also be fluorescently labelled with Cy2, Cy3, or Cy5 prior to 2DE [80] . CyDyes are cyanine dyes carrying an N-hydroxysuccinimidyl ester reactive group that covalently binds the e-amino group of lysine residues in proteins. During DIGE [81] , proteins in each of up to 3 samples can be labelled with one of these fluorescent dyes, and the differentially labelled samples can be mixed and loaded together on one single gel, allowing the quantitative comparative analysis of three samples using a single gel ( Figure 2 ). The DIGE technique has exhibited higher sensitivity as well as linearity, eliminated postelectrophoretic processing (fixing and destaining) steps and enhanced reproducibility by directly comparing samples under similar electrophoretic conditions [81, 82] . The resulting images are then analyzed by software such as De-Cyder which are specifically designed for 2D-DIGE analysis [83] . The major advantages of 2D-DIGE are the high sensitivity and linearity of its dyes, its straightforward protocol, as well as its significant reduction of intergel variability, increasing the possibility to unambiguously identify biological variability, and reducing bias from experimental variation. Moreover, the use of a pooled internal standard, loaded together with the control and experimental samples, increases quantification accuracy and statistical confidence [84] . The DIGE technique has dramatically improved the reproducibility, sensitivity, and accuracy of quantitation; however, its labeling chemistry has some limitations; proteins without lysine cannot be labeled, and they require special equipment for visualization, and fluorophores are very expensive [83, 85] . Tag (ICAT) . Gel-free, or MS based, proteomics techniques are emerging as the methods of choice for quantitatively comparing proteins levels among biological proteomes, since they are more sensitive and reproducible than two-dimensional gel-based methods. ICAT is one of the most employed chemical isotope labeling methods and the first quantitative proteomic method to be based solely on using MS [86, 87] . Each ICAT reagent consists of three essential groups: a thiol-reactive group, an isotope-coded light or heavy linker, and a biotin segment to facilitate peptide enrichment. In an ICAT experiment, protein samples are first labeled with either light or heavy ICAT reagents on cysteine thiols. The mixtures of labeled proteins are then digested by trypsin and separated through a multistep chromatographic separation procedure. Peptides are identified with tandem MS, and the relative quantifications of peptides are inferred from the integrated LC peak areas of the heavy and light versions of the ICAT-labeled peptides [88] . The ICAT concept has been widely used after its introduction [89] [90] [91] . Different software programs were developed to analyze ICAT labeled MS data (e.g., proICAT from Applied Biosystems, spectrum Mill from Agilent Technologies, and Sashimi from the Institute of System Biology [92] ). ICAT is extremely helpful to detect peptides with low expression levels, which is one of the bottleneck issues in analytic protein techniques [93, 94] . However, major limitations of this technique include selective detection of proteins with high cysteine content and difficulties in the detection of acidic proteins [95, 96] . The methods for direct comparison of DIGE and ICAT for the identification and quantification of proteins in complex biological mixtures are also being considered [97] .