Immunoaffinity Enrichment

Immunoaffinity enrichment is based on specific interaction of post-translationally modified site with an antibody (polyclonal or monoclonal) directed to the PTM epitope. Antibodies are immobilized, typically in bead format, to provide bait for capture of the PTM site of interest. Postenrichment, methylated, or acetylated peptides can be identified and characterized by LC-MS/MS. It has become routine to incorporate isotopic labels (e.g., SILAC, iTRAQ) or label-free quantitative approaches to compare PTM profiles across multiple biological conditions. Heavy methyl-SILAC and iMethyl-SILAC labeling strategies aid identification and reduce false positives [46, 98].

Antibodies recognizing methylated arginine in the context of an Arg-Gly motif have been proved effective for enriching modified proteins and peptides [39, 99, 100]. Pan-specific immunoprecipitation using panels of antibodies against lysine methylation [101] and specific antibodies against monomethyl- arginine, asymmetric dimethyl-arginine; monomethyl-, dimethyl-, and trimethyl-lysine motifs have been generated for comprehensive profiling of the “methylome” [46, 100]. Prior to this, there were concerns over the effectiveness of methylation-specific antibodies for pan enrichment as reviewed by Carlson and Gozani [102]. Similarly, a panel of seven antibodies of complementary specificities has been raised for Kac affinity enrichment of peptides [19], which follows a series of landmark studies directed to cataloging of Kac sites. Other studies have focused on immunoprecipitation at the protein level using antibodies against methylated (arginine, lysine) [102] or lysine-acetylated proteins [103].

There is increasing recognition of PTM cross talk, which can be addressed experimentally via a serial enrichment strategy for quantitative analysis of alterations in protein abundance and PTM profile [96]. In this workflow, termed SEPTM, the samples to be compared are labeled with either heavy or light SILAC reagents and trypsin digested to generate peptides. Sequential enrichment of phosphorylated, ubiquitylated, and acetylated peptides is achieved using a series of immobilized capture reagents, with the flow-through from each enrichment step used as the input for the subsequent step. Proof of concept to cataloging of multiple PTM has been generated for human leukemia cells cultured in the presence and absence of the proteasome inhibitor, bortezomib. Fractionation by basic pH reversed-phase HPLC was evaluated and provides partitioning of PTM and unmodified peptides to significantly improve the depth of coverage for Kac peptides relative to unfractionated samples [19, 96]. Interestingly, while the ubiquitinome altered in response to proteasomal inhibition, the acetylome was unaltered, but 414 of 1554 Kac sites occurred on lysine residues that were also identified as sites of ubiquitination - indicating the power of the method for parallel analysis of PTM in the same sample set to provide functional information on mechanisms of PTM interaction [96].

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