Improved enrichment strategies for phosphorylated peptides on titanium dioxide using methyl esterification and pH gradient elution

Abstract

Improvements to phosphopeptide enrichment protocols employing titanium dioxide (TiO2) are described and applied to identification of phosphorylation sites on recombinant human cyclin-dependent kinase 2 (CDK2). Titanium dioxide binds phosphopeptides under acidic conditions, and they can be eluted under basic conditions. However, some nonphosphorylated peptides, particularly acidic peptides, bind and elute under these conditions as well. These nonphosphorylated peptides contribute significantly to ion suppression of phosphopeptides and also increase sample complexity. We show here that the conversion of peptide carboxylates to their corresponding methyl esters sharply reduces nonspecific binding, improving the selectivity for phosphopeptides, just as has been reported for immobilized metal affinity chromatography (IMAC) columns. We also present evidence that monophosphorylated peptides can be effectively fractionated from multiply phosphorylated peptides, as well as acidic peptides, via stepwise elution from TiO2 using pH step gradients from pH 8.5 to pH 11.5. These approaches were applied to human CDK2 phosphorylated in vitro by yeast CAK1p in the absence of cyclin. We confirmed phosphorylation at T160, a site previously documented and shown to be necessary for CDK2 activity. However, we also discovered several novel sites of partial phosphorylation at S46, T47, T165, and Y168 when ion-suppressing nonphosphorylated peptides were eliminated using the new protocols.

Phosphopeptide enrichment using microscale titanium dioxide solid phase extraction

“…The different aspect ratios of SPE devices and load/wash volumes may have affected the recovery and selectivity of enrichment methods. The impact of loading and eluting conditions is not fully understood [34,35].…”

Recent Applications of Phosphoproteomics

“…Simon et al applied the methyl esterification on the carbocyclic-terminal of amino acid to deal with non-specific binding in titanium dioxide as it was quite efficient in IMAC and found that it also works effectively in this case. Together, they also showed the results of separation of monophosphorylated peptides and multi-phosphorylated peptides by gradient elution from pH8.5 to pH11.5 [26]. Li et al demonstrated that the ratio of loading sample amount to titanium dioxide is also an important factor for specificity of enrichment [27].…”

Enrichment strategies for phosphoproteomics: state-of-the-art

“…Using high concentration during the binding step may improve specifi city, but more weakly bound phosphopeptides could be lost, especially monophosphorylated peptides. Another approach how to minimize the level of nonspecifi cally bound peptides is chemical derivatization before the enrichment, e.g., methylesterifi cation of peptides (Ficarro et al 2002, Simon et al 2008. A variety of pH conditions during the binding or the elution step have been employed to optimize the protocol for enrichment on MOAC resins (Simon et al 2008 , Park andMaudsley 2011 ).…”