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