Tim Wehr

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 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.