Comparing Multistep Immobilized Metal Affinity Chromatography and Multistep TiO₂ Methods for Phosphopeptide Enrichment

Abstract

Phosphopeptide enrichment from complicated peptide mixtures is an essential step for mass spectrometry-based phosphoproteomic studies to reduce sample complexity and ionization suppression effects. Typical methods for enriching phosphopeptides include immobilized metal affinity chromatography (IMAC) or titanium dioxide (TiO2) beads, which have selective affinity and can interact with phosphopeptides. In this study, the IMAC enrichment method was compared with the TiO2 enrichment method, using a multi-step enrichment strategy from whole cell lysate, to evaluate their abilities to enrich for different types of phosphopeptides. The peptide-to-beads ratios were optimized for both IMAC and TiO2 beads. Both IMAC and TiO2 enrichments were performed for three rounds to enable the maximum extraction of phosphopeptides from the whole cell lysates. The phosphopeptides that are unique to IMAC enrichment, unique to TiO2 enrichment, and identified with both IMAC and TiO2 enrichment were analyzed for their characteristics. Both IMAC and TiO2 enriched similar amounts of phosphopeptides with comparable enrichment efficiency. However, phosphopeptides that are unique to IMAC enrichment showed a higher percentage of multi-phosphopeptides, as well as a higher percentage of longer, basic, and hydrophilic phosphopeptides. Also, the IMAC and TiO2 procedures clearly enriched phosphopeptides with different motifs. Finally, further enriching with two rounds of TiO2 from the supernatant after IMAC enrichment, or further enriching with two rounds of IMAC from the supernatant TiO2 enrichment does not fully recover the phosphopeptides that are not identified with the corresponding multi-step enrichment.

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“…Our previous work demonstrated that multiple rounds of enrichment, performed sequentially, provided efficient enrichment and recovery of phosphopeptides [15, 16],. Three rounds of IMAC enrichment yielded 4,331 phosphopeptides, 30% of the total identifications.…”

“…Phosphopeptides were enriched using both IMAC and TiO 2 beads to enhance coverage, since the two chemistries have been reported to enrich complementary sub-proteomes [14, 15]. Digests were performed in triplicate on two occasions, once for each enrichment method.…”

“…Digests were performed in triplicate on two occasions, once for each enrichment method. IMAC was performed three times sequentially, with the second and third enrichments mixed together, as described by us previously [15, 16]. Briefly, desalted peptides from 3.5mg of lysate were resuspended in loading buffer (40% ACN, 25mM formic acid in water) and mixed with 30uL of prepared PHOS-Select IMAC beads (Sigma).…”

“…TiO 2 was performed twice as previously reported [15, 17]. Bulk 5 µm Titanosphere particles were purchased from GL Sciences USA (Torrance, CA).…”

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“…51 For example, Fe 3+ and Ga 3+ -based IMAC strategies are often biased toward enrichment of multiply phosphorylated peptides, while TiO 2 -based chromatography is typically biased toward mono-phosphorylated peptides. 49, 52, 53 Physiological loading buffers such as PBS and certain additives to prevent non-specific binding of acidic peptides (e.g. citric acid) have also been shown to bias enrichments toward multiply phosphorylated peptides, presumably because peptides with several phosphorylation groups have stronger metal ion affinity.…”