Immobilized Metal Affinity Chromatography (IMAC) Service
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Immobilized metal affinity chromatography (IMAC) is another strategy for enriching phosphorylated peptides before LC-MS/MS. It uses positively charged ions, commonly Fe3+, to capture negatively charged phosphopeptides.
Comparison of PTMScan® antibody-based and IMAC phosphopeptide enrichment: PTM profiling of human gastric carcinoma (MKN-45) cells.
Phosphopeptides were enriched using various classes of motif/PTM antibodies (tyrosine, proline, serine/threonine, and basophilic), or IMAC methods.
- Over 20,000 phosphorylation sites were identified across all samples.
- There was minimal overlap between phosphopeptides enriched using antibody versus IMAC methods.
- The methods are highly complementary. For optimal coverage of the phosphoproteome use of both antibody-based and IMAC methods is recommended.
Combinations of motif antibodies were used to enrich phosphopeptides in MKN-45 cells as indicated (A). Area proportional Venn diagram of the overlap between antibody and IMAC enrichment of phosphopeptides from MKN-45 cells. Comparisons were made on unique proteins/sites (B).
View complete dataset showing complementarity of antibody and IMAC enrichment methods.
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