Strategies Relying on Chemical Derivatization

Besides the nonmodifying purification procedures described so far, methods exist that make use of chemical derivatization strategies for the enrichment of phosphorylated proteins and peptides. Two main approaches have been described. The first one consists of j elimination followed by Michael addition, where the phosphate group of pSer or pThr (pTyr cannot be derivatized using this method) is replaced with a nucleophile that is suitable for selective enrichment [15-21]. Alternative strategies involve temporary carbodiimid coupling of the phosphate residues of pSer/pThr/pTyr to a solid phase, followed by washing steps to remove nonphosphorylated species [22, 23]. However, due to the involved chemical processes, unwanted side reactions or may occur. This is known for the approach involving j elimination, which is carried out at basic conditions (pH 12-14). These reaction conditions can lead to the replacement of O-glycosides [24, 25], sulfonated residues [26], and even of hydroxyl groups, which are located on Ser and Thr [27]. Several other artificial protein modifications are known to occur under highly basic conditions [28]. The carbodiimid approach has the distinct advantage of including pTyr in the analysis and that no side reactions are reported so far. The initial approach [27] involved several modification steps which increases the likelihood of sample loss and modification in each step. The refined method [23, 29] involves methylesterification, which has also its drawbacks as described above (see IMAC section). Nevertheless, the method seems to be promising and might find its way into plant phosphorylation research.

IMAC. The most widespread method used for phosphopeptide enrichment is IMAC. This technique was initially developed by Porath et al. [30] and was originally used to separate all kinds of different proteins. It mainly relies on the attraction of a negatively charged amino acid residue to a positively charged metal that is immobilized on a metal chelator matrix such as iminodiacetic acid. Iron and Ga3+ are the most widely used metals for the enrichment of phosphorylated species. Since a phosphate group has a stronger net negative charge than any other amino acid residue, phosphorylated proteins/peptides are better retained on the matrix than their nonphosphorylated counterparts. IMAC has been used to separate plant-derived phos-phorylated proteins [31] as well as phosphopeptides [32, 33] from non-phosphorylated ones. However, there are some difficulties when applying IMAC. Histidine and Asp phosphorylations are not accessible by classic IMAC enrichment because they are acid-labile and IMAC loading is usually conducted under acidic conditions. IMAC has in fact been used to enrich for acidic proteins and is prone to unspecific binding of proteins/peptides rich in glutamic and aspartic acid residues [34, 35]. To circumvent the unspecific binding problem, peptides can be methylesterified, thus converting glutamic and aspartic acid residues into their noncharged methyl esters. Unfortunately, this reaction is conducted under harsh conditions (exclusion of water at pH 0-1) and is not free of side reactions such as deamination of glutamines and asparagines [36], making this method of methylesterification especially unsuitable for complex protein mixtures. However, it seems that the success of IMAC and methylesterification also depends on the kind of protein sample and IMAC resin used [37].

Since IMAC has been used for the enrichment of phosphorylated proteins [38] and peptides, it can in principle be applied to couple the advantages of both strategies. However, probably due to experimental difficulties, there is only little literature describing a double enrichment on both the protein and peptide level. A recent description comes from Collins et al. [39], who published a detailed protocol for this sort of sequential purification based on IMAC, and an analogous approach is described by Huang et al. [40]. There is no study describing this or a similar experimental setup for plant proteins. Nevertheless, recent advances in method refinement [see reference 41] might help to overcome some of the aforementioned difficulties of IMAC.

MOAC. Other inorganic approaches make use of metal oxides and hydroxides. This includes the recently developed methods based on zirconia, titania, and aluminum hydroxide (Al(OH)3). Since oxides and hydroxides are closely related and in fact all of these compounds belong to the oxide class of minerals [see reference 42], the general term metal oxide affinity chromatography may be used for these methods in analogy to IMAC. An example is shown in Figure 29.1.

Titania and Zirconia. Recently, it was shown that metal oxides can also be used for the enrichment of phosphorylated peptides. In these studies, affinity chro-matography based on titania (titanium dioxide, TiO2) [43-52] and zirconia (ZrO2) [53] was used. On-line coupling of a titania precolumn and an anion exchange [49, 50] or RP column [46, 48] in an HPLC setup has been shown to be useful in the selective analysis of phosphorylated peptides derived from proteolytic digests. Identification of the phosphopeptides was achieved by monitoring the UV trace [49, 50] or by using a mass spectrometer [46, 48]. Similarly, nanoparticles composed of Fe3O4/TiO2 core shell particles were used to specifically isolate and detect phosphopeptides [51]. Unspecific binding, which is also reported when using titania [46, 48], can be reduced by methylesterification [48] and the use of appropriate incubation buffers [52]. However, the use of a special incubation buffer is preferable because of the side reactions occurring during methylesterification (see above). While all of the studies named so far focused on the analysis of standard proteins or animal tissue, a very recent application comes from the plant field. In this study the authors used titania to enrich phospho-peptides of spinach stroma membranes and identified some new phosphorylation sites in photosynthesis-related proteins [53].

Aluminum Oxide and Hydroxide. Al(OH)3 is a well-known and widely used adjuvant in medicine. Adjuvants are additives that enhance the effectiveness of medical treatment by potentiating the immune response and functioning as a carrier for antigens [54, 55]. Because of this wide application, protein adsorption to Al(OH)3 has been studied in considerable detail and some of those studies also investigate the binding behavior of phosphorylated proteins to Al(OH)3 [56-59]. In addition, aluminium oxide, a close relative of Al(OH)3, has been reported to exhibit a high and selective attraction to phosphorylated biomolecules [60]. The conclusion of these studies is that Al(OH)3 and oxide both have a high affinity for phosphorylated biomolecules and

FIGURE 29.1. Phosphoprotein changes of A. thaliana after chemical treatment combined with MOAC enrichment: (A) Phosphoproteins of A. thaliana cell cultures without treatment. (B) Phosphoproteins of A. thaliana after 24 hr 2-deoxyglucose treatment, changes are indicated by white arrows. Proteins are analyzed by LC-MS/MS. Phosphoproteins were visualized with Pro-Q diamond stain. (Chen and Weckwerth, unpublished data.)

FIGURE 29.1. Phosphoprotein changes of A. thaliana after chemical treatment combined with MOAC enrichment: (A) Phosphoproteins of A. thaliana cell cultures without treatment. (B) Phosphoproteins of A. thaliana after 24 hr 2-deoxyglucose treatment, changes are indicated by white arrows. Proteins are analyzed by LC-MS/MS. Phosphoproteins were visualized with Pro-Q diamond stain. (Chen and Weckwerth, unpublished data.)

that the affinity towards, phosphate is considerably higher than for sulfate or nitrate. These characteristics make them good candidates for the enrichment of phosphorylated proteins and peptides out of complex mixtures. However, it is also clear from some of those studies that nonphosphorylated proteins also exhibit a considerable affinity to those matrices even if they are not phosphorylated.

The enrichment procedure is usually performed under denaturing conditions to decrease the threat of dephosphorylation and degradation events because of the action of endogenous proteases and phosphatases [61, 62] Proteins are extracted using a mix of buffer, phenol, NaF (which serves as a phosphatase inhibitor), and 2-ME. After precipitation, proteins are resuspended in a special incubation buffer containing urea, subjected to affinity chromatography, and eluted using pyrophosphate. So far, over 300 putative phosphoproteins from different sources including C. reinhardtii, A. thaliana cell cultures, seeds, and leaves have been enriched (Wolschin and Weckwerth, unpublished results).

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