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

Abstract

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 Ga(3+) and Fe(3+) for phosphopeptides, we designed a metal-directed immobilized metal ion affinity chromatography for the sequential enrichment of phosphopeptides. In Raji B cells, the sequential Ga(3+)-Fe(3+)-immobilized metal affinity chromatography (IMAC) strategy displayed a 1.5-3.5-fold superior phosphoproteomic coverage compared to single IMAC (Fe(3+), Ti(4+), Ga(3+), and Al(3+)). In addition, up to 92% of the 6283 phosphopeptides were uniquely enriched in either the first Ga(3+)-IMAC (41%) or second Fe(3+)-IMAC (51%). The complementary properties of Ga(3+) and Fe(3+) were further demonstrated through the exclusive enrichment of almost all of 1214 multiply phosphorylated peptides (99.4%) in the Ga(3+)-IMAC, whereas only 10% of 5069 monophosphorylated phosphopeptides were commonly enriched in both fractions. The application of sequential Ga(3+)-Fe(3+)-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 phosphorylation sites were predominately enriched in the first Ga(3+)-IMAC (72%), while Pro-directed (85%) and basophilic (79%) phosphorylation sites were enriched in the second Fe(3+)-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 Ga(3+)-Fe(3+)-IMAC, we propose that this strategy allows more comprehensive characterization of the phosphoproteome both in vitro and in vivo.

Preparation of on-plate immobilized metal ion affinity chromatography platform via dopamine chemistry for highly selective isolation of phosphopeptides with matrix assisted laser desorption/ionization mass spectrometry analysis

“…In Tsai's work, a tip was packed with Ni-NTA silica resin and immobilized multimetal on it to enrich phosphopeptides, which could avoid the above problems mentioned. However, it was hard for it to realize high throughput analysis, and it also needed to elute during the process of phosphopeptides enrichment like other off-target methods [14]. To avoid these problems, on-target isolation prior to MALDI-TOF-MS analysis has been developed and attracted much attention recently, which involves in on-plate MOAC platform for the selective isolation of phosphopeptides.…”

Liquid phase based separation systems for depletion, prefractionation, and enrichment of proteins in biological fluids and matrices for in‐depth proteomics analysis—An update covering the period 2011–2014

“…Tsai et al [62] designed a metal-directed IMAC for the sequential enrichment of phosphopeptides. Using Raji B cells, the sequential Ga 3+ -, Fe 3+ -IMAC method displayed a much better detection sensitivity compared to the use of a single IMAC (Fe 3+ , Ti 4+ , Ga 3+ , and Al 3+ ).…”

Recent advances in phosphoproteomics and application to neurological diseases

“…54 Thus, the combination of different metal ions or approaches has been used to achieve wider coverage of the whole phosphoproteome. 55, 56 …”