Elsevier

Analytical Biochemistry

Volume 377, Issue 2, 15 June 2008, Pages 234-242
Analytical Biochemistry

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

https://doi.org/10.1016/j.ab.2008.03.024Get rights and content

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.

Section snippets

Materials

Triethylammonium bicarbonate (TEAB), DHB, iodoacetamide, dithiothreitol, 3-indoleacrylic acid, and β-casein (bovine) were purchased from Sigma–Aldrich (St. Louis, MO, USA). TiO2 particles (50 μm) were purchased from Glygen (Columbia, MD, USA). Sequencing-grade modified trypsin was purchased from Promega (Madison, WI, USA). NH4OH, trifluoroacetic acid (TFA), high-performance liquid chromatography (HPLC)-grade water, and acetonitrile (ACN) were purchased from Fisher Scientific (Fairlawn, NJ, USA).

pH-dependent elution for fractionation of monophosphorylated peptides from multiply phosphorylated peptides

Larsen and coworkers emphasized that elution buffers of pH > 10.5 were necessary to efficiently elute multiply phosphorylated peptides from TiO2[19]. This suggested that fractionation (in addition to enrichment) of monophosphorylated peptides from multiply phosphorylated peptides would be possible by eluting sequentially from low to high pH. We explored this possibility using a two-step elution strategy. Peptides were eluted from TiO2 initially with 100 mM TEAB (pH 8.5) followed by a subsequent

Discussion

Greater selectivity for enrichment of phosphopeptides on TiO2 supports can be attained by moderating buffer pH (for monophosphorylated peptides) and methyl esterification. These methods were employed in the characterization of CDK2 phosphorylation by yeast CAK1p in the absence of cyclin. We initially evaluated the protocol developed by Larsen and coworkers [19] that used DHB as an additive to deter nonspecific binding to TiO2 and elution with NH4OH (pH > 10.5). We found that nonspecific binding

Acknowledgments

The authors thank John Strahler, Angela Walker, and Maureen Kachman of the Michigan Proteomics Consortium for their helpful insight and discussion. This work was supported by National Institutes of Health (NIH)/National Center for Research Resources (NCRR), National Resource for Proteomics and Pathways grant P41-18627 to P.C.A.

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