PTMs of proteins are considered to be one of the major determinants regarding organisms complexity [201] . To date, at least more than 200 different types of PTMs have been identified of which only a few are reversible and important for the regulation of biological processes. Specific functions are usually mediated through PTMs, such as phosphorylations, acetylations, or glycosylations, which places additional demands on the sensitivity and precision of the method [202] . One of the most studied PTMs is protein phosphorylation, because it is vital for a large number of protein functions that are important to cellular processes spanning from signal transduction, cell differentiation, and development to cell cycle control and metabolism. Enzymes and receptors can be switched "on" and "off " by phosphorylation and dephosphorylation. It was estimated that 10-50% of proteins are phosphorylated. Phosphorylation often occurs on serine, threonine, and tyrosine residues in eukaryotic proteins [203] . Analysis of the entire cellular phosphoproteome has been an attractive study subject since the discovery of phosphorylation as a key regulatory mechanism of cell life. Unfortunately, phosphoproteins analysis is not straightforward for five main reasons. First, the stoichiometry of phosphorylation is generally relatively low, because only a small fraction of the available intracellular pool of a protein is phosphorylated at any given time as a result of a stimulus. Second, the phosphorylatation sites on proteins might vary, implying that any given phosphoprotein is heterogeneous (i.e., it exists in several different phosphorylated forms). Third, many of the signaling molecules, which are major targets of phosphorylation events [204] , are present at low abundance within cells and, in these cases; enrichment is a prerequisite before analysis. Fourth, most analytical techniques used for studying protein phosphorylation have a limited dynamic range, which means that although major phosphorylation sites might be located easily, and minor sites might be difficult to identify. Finally, phosphatases could dephosphorylate residues unless precautions are taken to inhibit their activity during preparation and purification steps of cell lysates. In addition, various methods for protein phosphorylation site determination have been developed, yet this task remains a technical challenge [205] . Western blot has been widely used to determine the presence of PTMs. However, this technique relies on the prior knowledge of the type and position of specific modifications and the availability of antibodies. It has low throughput and not ideal for studying highly complicated samples. Specific chemical or affinity enrichment steps are usually incorporated into the sample preparation or fractionation stages of the general scheme of proteomic studies [206, 207] . Well established methods involving the analysis of 32P-labeled phosphoproteins by Edman degradation and two-dimensional phosphopeptide mapping have proven to be powerful but not without limitations. Consequently, mass spectrometry (MS) has emerged as a reliable and sensitive method for the characterization of protein phosphorylation sites [208] and may therefore represent a method of choice for the analysis of protein phosphorylation [209] . Immobilized metal affinity chromatography (IMAC), Metal oxide affinity chromatography (MOAC), and covalent methods are all capable of selectively enriching phosphopeptides [210] . MOAC based on adsorption to TiO2 is especially attractive, but as with all techniques, loading, rinsing, and elution solutions must be carefully selected to minimize nonspecific adsorption and to maximize the detection of both monophosphorylated and multiphosphorylated species. IMAC might not provide the selectivity available with TiO2 enrichment, but with appropriate reagents, IMAC can be selective and sensitive for monophosphorylated and tetraphosphorylated peptides. However, some buffers and reagents such as EDTA are not compatible with IMAC, so HPLC purification may be needed prior to this technique [211] . When trying to isolate and identify as many phosphoproteins as possible in a cell lysate, chromatographic column-based methods are required. Multiple elutions from IMAC or MOAC columns or even gradient elutions can help to simplify fractions of proteins and reveal more peptides [212, 213] . A combination of techniques can reveal large numbers of phosphopeptides in complex samples, but comprehensive phosphoproteomics is still not possible. For the highest protein coverage, future phosphoproteomic techniques will likely employ multiple enrichment techniques along with two-dimensional separations, but such studies are time consuming. Combinations of affinity-based enrichment and extraction methods, multidimensional separation technologies, and mass spectrometry are particularly attractive for systematic investigation of posttranslationally modified proteins in proteomics [214] .