Tim Wehr
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The reversible phosphorylation of specific sites on proteins is implicated in the control of multiple cellular functions and
processes, including cell growth and differentiation, cell death, gene expression, and signal transduction. Almost a third
of all cellular proteins are believed to be phosphorylated at any given point in the cell cycle. Given the importance of protein
phosphorylation in cell activities, characterization of the phosphoproteome will be key to understanding the mechanism of
cellular processes and in identifying targets for therapeutic intervention. However, phosphoproteome analysis is a daunting
analytical challenge. Phosphorylation is dynamic, with many proteins being phosphorylated at different sites and at different
times. Therefore, phosphorylation at a given site can be substoichiometric and, thus, in very low abundance. Currently, the
dominant technologies for characterizing protein phosphorylation are matrix-assisted laser desorption ionization–time of flight
(MALDI-TOF) mass spectrometry (MS) and liquid chromatography (LC)–nanoelectrospray tandem MS. Most MS-based protein characterization
is performed at the peptide level after cleavage with site-specific proteases. Phophorylation sites are less than ideal candidates
for these approaches. Phosphorylated peptides do not ionize well in positive ion mode (the preferred approach in most cases),
and are subject to ion suppression in the presence of the great excess of nonphosphorylated peptides. The most effective strategy
for phosphoproteome characterization is an initial enrichment to extract phosphoproteins or phosphopeptides from the bulk
proteome. There are a variety of enrichment methods, including immunoprecipitation, chemical derivatization, immobilized metal
affinity chromatography (IMAC), and enrichment on metal oxide surfaces. This installment of "Directions in Discovery" will
review the current approaches, compare their performance, and take a look at some recent enrichment technologies.
Immunoprecipitation
Immunoprecipitation of proteins with antibodies specific to phosphorylated residues is an obvious strategy for enrichment
of phosphoproteins from complex mixtures (1,2). This approach has been quite successful in the enrichment of proteins phosphorylated
at tyrosine residues (3), as high-quality antiphosphotyrosine antibodies that have little crossreactivity with other phosphorylated
or nonsposphorylated residues are available. In this application, immunoprecipitation is particularly valuable because phosphorylation
at tyrosine is relatively rare, accounting for a small fraction of the phosphorylation sites. Antiphosphotyrosine antibodies
generally are not considered suitable for the enrichment of phosphotyrosine-containing peptides because of poor selectivity
(4). However, Rush and colleagues (5) successfully enriched phophopeptides from trypsin-digested cell lysates using antiphosphotyrosine
antibody immobilized on agarose beads. Zhang and Neubert (4) demonstrated that the selectivity of immunoprecipitation of tyrosine
phosphopeptides from digests of cell lysates can be improved significantly by the addition of the detergent n-ocylglucoside to the immunoprecipitation buffer. To date, no antibodies suitable for enrichment of proteins containing phosphoserine
or phosphothreonine residues are available.
Chemical ModificationChemical modification strategies for the enrichment of phosphoproteins or phosphopeptides rely upon the chemical properties
of the phosphate groups. Two approaches have been described. One uses β-elimination of the phosphate group to create a site
for introduction of an affinity ligand. The other uses conversion of the phosphate group to a phosphoramidate to facilitate
enrichment of phosphopeptides on affinity supports. Chemical modification procedures can be very selective for phosphate enrichment,
but their complexity can compromise overall recovery, and side reactions can complicate interpretation of results.
Beta Elimination With Chemical Modification
Under strongly alkaline conditions, phosphoserine and phosphothreonine lose H3PO4 via a β-elimination to yield dehydroalanine and dehydroaminobutyric acid, respectively. The Chait laboratory (6) employed
this chemistry as a means of introducing a group for affinity enrichment by a Michael addition to the newly formed double
bond (Figure 1). In their initial approach, proteins were first treated with performic acid to oxidize cysteine, cystine,
and methionine residues. Then the β-elimination was performed under alkaline conditions, with ethanedithiol added to modify
the double bond. Following ethanedithiol addition, the resultant free thiol was used as the attachment point for a biotin
affinity tag using an alkylating agent linked to biotin. Tagged proteins were enriched on a monomeric avidin column, then
eluted for MS analysis. This approach was limited by the inefficient recovery of tagged peptides from the affinity column,
and instability of portions of the affinity tag under MS fragmentation conditions.
The β-elimination strategy was improved later by replacing the biotin affinity tag with an activated thiol affinity resin
(7). Following β-elimination and Michael addition with ethanedithiol or dithiothreitol, modified peptides were captured via
disulfide exchange on the activated thiol affinity column, then eluted with dithiothreitol.
The improved protocol increased sensitivity from nanomole to subpicomole levels. However, the method suffers from two limitations.
First, a small proportion (1–2%) of nonphosphorylated serine residues undergoes β-elimination. This side reaction can be minimized
by using dithiothreitol in place of ethanedithiol for the addition reaction, and by reducing the concentration of base used
for the elimination reaction. The addition of ethylene diamine tetraacetic acid (EDTA) to chelate trace metals reduces the
side reaction 10-fold, but also reduces the reaction efficiency of phosphopeptides. The second limitation of this enrichment
strategy is the conversion of O-glycosylated serines to dehydroalanine under the enrichment conditions.
