Highly Specific Capture and Direct MALDI MS Analysis of Phosphopeptides by Zirconium Phosphonate on Self-Assembled Monolayers

Abstract

The dynamic range and low stoichiometry of protein phosphorylation frequently demands the enrichment of phosphorylated peptides from protein digests prior to mass spectrometry. Several techniques have been reported in literature for phosphopeptide enrichment, including metal oxides such as TiO(2) and ion metal affinity chromatography (IMAC). While the metal oxides have been used with reasonable success, IMAC has suffered from reduced selectivity and poor reproducibility. In this report, we present the first demonstration of the use of immobilized zirconium on a phosphonate-terminated self-assembled monolayer (SAM) for specific phosphopeptide capture and direct analysis by MALDI MS. By using the herein described functionalized-surface-based technology, efficient enrichment of phosphopeptides in different standard test systems such as alpha- or beta-casein digests or synthetic phosphopeptides spiked in nonphosphorylated protein digest has been demonstrated. The limit of detection for a beta-casein phosphopeptide was assessed to be at the low femtomole level. Compared to other state-of the-art technologies, like use of TiO(2) and Fe-IMAC, the presented technique demonstrated a superior performance with respect to specificity and bias with respect to singly or multiply phosphorylated peptides. Additionally, this platform was also successfully applied for ESI sample preparation, providing detailed sequence information of the investigated phosphopeptide. This technology was also proven to be applicable for real life samples such as phosphorylation site analysis of recombinant human MAPK1 and HSP B1 isolated from a 2D-gel spot by phosphopeptide enrichment and direct MALDI MS/MS.

Nanoparticle-based immunosensor with apoferritin templated metallic phosphate label for quantification of phosphorylated acetylcholinesterase

“…Numerous studies have shown that ZrO 2 NPs have strong affinity to OP pesticides (Liu et al, 2008;Du et al, 2008a,b), phosphoric peptides (Hoang et al, 2010;Kweon and Håkansson, 2006) through chelation with phosphoric group. Motivated by these results, ZrO 2 NPs were employed here as capture reagent to recognize phosphorylated AChE.…”

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“…It is inorganic oxide, whose band gap estimates from 5 to 7 eV [17,18] depending on the phase (cubic, tetragonal, monoclinic, or amorphous) and preparation methods. Researchers have also demonstrated that ZrO 2 has a strong affinity to negatively charge moieties [19], which motivated us to use graphene oxide (GO)-ZrO 2 nanocomposite as selective photocatalyst for heavy metals. Moreover, various papers have been published recently on the graphene-ZrO 2 nanocomposites applications, which include the deoxyribonucleic acid sensing detection [20], organophosphorus agents [21], supercapacitor energy-storage application [22], and photocatalytic degradation of antimonite (Sb(III)) and antimonate (Sb(V)) from aqueous solution [23].…”

An ICP-MS, ESI-MS and molecular modelling investigation of homogeneous gallium affinity tagging (HMAT) of phosphopeptides

“…[15][16][17][18][19][20][21][22][23][24][25][26][27][28][29] Many different analytical techniques have been developed to specifically enrich phosphopeptides including Immobilised Metal Affinity Chromatography (IMAC), utilizing iron, gallium, and more recently titanium and zirconium. [15,18,[29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45][46][47] The efficiency of each of these metals in selectively binding phosphopeptides has been extensively investigated many times with varying success. This has been attributed to the non-specific binding of the acidic amino acid residues asparagine and glutamine to the metal ions, the binding of hydrophobic residues to the gel bed and the elution of multiply phosphorylated peptides and impurities in the metal oxide material.…”