Phosphorylation is one of the most frequently occurring postranslational modifications (almost 30% of all proteins are thought to be phosphorylated). In Eukaryotes, regulation of cellular processes is achieved through reversible phosphorylation of receptors, adaptor proteins and protein kinases on .  It plays an important role in signal transduction and regulates diverse cellular processes such as growth, metabolism, proliferation, motility and differentiation.

Analysis of the entire complement of phosphorylated proteins in cells or the “phosphoproteome” has become a realistic goal due to new enrichment protocols for phosphoproteins and phosphopeptides and improvement of methods to selectively visualize phosphorylated residues by mass spectrometry. 

Procedures to identify phosphorylated proteins

1) Traditional methods include 

• radioactive labeling with 32P-labeled ATP followed by  , 

• Edman sequencing and 

• phosphospecific antibodies. 

Each of these techniques exhibits shortcoming for quantitative proteome analysis. For example, labeling with 32P requires a viable cell source and therefore is not applicable for the proteomic analysis of human tissue specimens. Most traditional methods are also inadequate because it is impossible to obtain large amounts of proteins since most signaling molecules are not abundantly expressed and their stoichiometry of phosphorylation is quite low (phosphoproteins are often a small fraction of the individual protein concentration). 

2) Mass Sectrometry has become the technique of choice for phosphorylation analysis. There are, however, the following challenges:

• ion signals corresponding to phosphorylated peptides are significantly suppressed in the presence of non-phosphorylated peptides. Thus purification steps to enrich phosphorylated proteins (discussed below) from non-phosphorylated proteins or phosphopeptides from non-phosporylated peptides is usually necessary. 

• Phosphopeptides are negatively charged whereas electrospray MS is run in the positive mode

• Phosphopeptides are hydrophilic and do not bind well to usual prep columns (like C18 discussed below)

• are labile (B-elimination), whereas phosphotyrosine is relatively stable

Enrichment of phosphoproteins

(1) By Chromatography

• Miniaturized reverse-phage C18 columns: The problem here is that phosphopeptides are hydrophilic which results in losses.

• Polymer-based reverse-phage (oligo R3) and porous graphite carbon (PGC): have a much higher capacity for phosphopeptides and hydrophilic peptides than C18 columns

• Immobilized metal affinity chromatography (IMAC): is based on the affinity of negatively charged phosphate groups for positively charged metal ions (e.g. Fe3+) immobilized on a chromatographic support. The major limitation of this method is non-specific binding of non-phosphorylated peptides containing residues possessing acidic side chains such as  . Strategies to overcome this problem is the derivatization of all peptides by a methyl esterification reaction that reduces non-specific binding by carboxylate groups. 

(2) By Phosphospecific Antibodies: This is the simplest method to enrich phosphoproteins. Although there are several commercially available antibodies for phosphorylated (not good for phosphopeptides, however), there are currently no suitable antibodies for phosphorylated .

(2) By Chemical Modification:

• Alkaline B-elimination: under strongly alkaline conditions, phosphoserine and phosphothreonine residues undergo an elimination reaction whereby phosphoric acid is lost and an alpha, beta unsaturated bond is formed. The end products can be detected by tandem   (MS/MS). 

• chemical modification based on B-elimination: Here, samples containing phosphoproteins are first treated with a strong base, leading to B-elimination reaction in the case of phosphoserine and phosphothreonine residues. A reactive species containing an alpha, beta unsaturated bond is formed. The biotinylated reagent reacts with sulfhydryl (SH) groups. Biotinylated phosphoprotein is now tagged for enrichment on avidin columns in later steps.

Some advantages here is that the avidin-biotin extraction results in phosphopeptide enrichment.

Some possible disadvantages of this method is that glycosyl groups on Ser and Thr will be eliminated giving positive results. Thus deglycosylation will be required. 

• carbodiimide condensation reaction: A disadvantage here is that the multistep chemical procedure requires 13 hours and has low yield. There are also side reactions that may interfere.

Quantitative Phosphoproteomics

There are several reasons why quantitation of phosphorylation is important. For example, the ratio of phosphorylation of a protein on multiple residues might be crucial for its function. MS-based quantitation techniques are emerging.

In one scheme of quantitation of phosphopeptides using , 2 states of phosphopeptides are labeled with isotopically distinct biotinylated mass tags. After labeling, the samples are mixed and purified over an avidin column. The unbound peptides are removed by washing followed by elution of tagged peptides and analysis by MS. The mass spectrum shows pairs of peaks for formerly phosphorylated peptides owing to the mass difference introduced by biotin tags of 2 different masses. The relative amounts of phosphopeptides in the 2 states can be derived from the relative intensities of the two peaks.

In another scheme, two protein pools differing in their extent of phosphorylation are digested with trypsin either in H216O or in H218O to obtain differential mass labeling. Equal amounts of the two pools are mixed, and phosphopeptides are selected with IMAC beads charged with Fe3+. The two peptide pools can be distinguished by a shift of 4 Da in the isotope cluster, and the difference in the extent of phosphorylation is reflected by the peak areas of the two monoisotopic peaks.