Sequential Phosphoproteomic Enrichment through Complementary Metal-Directed Immobilized Metal Ion Affinity Chromatography

Methodologies to enrich heterogeneous types of phosphopeptides are critical for comprehensive mapping of the Under-explored phosphoproteome. Taking advantage of the distinct binding affinities of Ga3+ and Fe3+ for phosphopeptides, we designed a metal-directed immobilized metal ion affinity chromatography for the sequential enrichment of phosphopeptides. In Raji B cells, the sequential Ga3+-Fe3+ -immobilized metal affinity chromatography (IMAC) strategy displayed a 1.5-3.5-fold superior phosphoproteomic coverage compared to single IMAC (Fe3+, Ti4+, Ga3+, and Al3+). In addition, up to 92% of the 6283 phosphopeptides were uniquely enriched in either the first Ga3+-IMAC (41%) or second Fe3+-IMAC (51%). The complementary properties of Ga3+ and Fe3+ were further demonstrated through the exclusive enrichment of almost all of 1214 multiply phosphorylated peptides (99.4%) in the Ga3+ -IMAC, whereas only 10% of 5069 monophosphorylated phosphopeptides were commonly enriched in both fractions. The application of sequential Ga3+ -Fe3+-IMAC to human lung cancer tissue allowed the identification of 2560 unique phosphopeptides with only 8% overlap. In addition to the above-mentioned mono- and multiply phosphorylated peptides, this fractionation ability was also demonstrated on the basic and acidic phosphopeptides: acidophilic phosphorylationv sites were predominately enriched in the first Ga3+-IMAC (72%), while Pro-directed (85%) and basophilic (79%) phosphorylation sites were enriched in the second Fe3+-IMAC. This strategy provided complementary mapping of different kinase substrates in multiple cellular pathways related to-cancer invasion and metastasis of lung cancer. Given the fractionation ability and ease of tip preparation of this Ga3+-Fe3+-IMAC, we propose that this strategy allows more comprehensive characterization of the phosphoproteome both in vitro and in vivo.