Automated, reproducible, titania-based phosphopeptide enrichment strategy for label-free quantitative phosphoproteomics

Abstract

An automated phosphopeptide enrichment strategy is described using titanium dioxide (TiO2)-packed, fused silica capillaries for use with liquid chromatography (LC)-mass spectrometry (MS)/MS-based, label-free proteomics workflows. To correlate an optimum peptide:TiO2 loading ratio between different particle types, the ratio of phenyl phosphate-binding capacities was used. The optimum loading for the column was then verified through replicate enrichments of a range of quantities of digested rat brain tissue cell lysate. Fractions were taken during sample loading, multiple wash steps, and the elution steps and analyzed by LC-MS/MS to gauge the efficiency and reproducibility of the enrichment. Greater than 96% of the total phosphopeptides were detected in the elution fractions, indicating efficient trapping of the phosphopeptides on the first pass of enrichment. The quantitative reproducibility of the automated setup was also improved greatly with phosphopeptide intensities from replicate enrichments exhibiting a median coefficient of variation (CV) of 5.8%, and 80% of the identified phosphopeptides had CVs below 11.1%, while maintaining >85% specificity. By providing this high degree of analytical reproducibility, this method allows for label-free phosphoproteomics over large sample sets with complex experimental designs (multiple biological conditions, multiple biological replicates, multiple time-points, etc.), including large-scale clinical cohorts.

Keywords: glycolic acid; liquid chromatography; mass spectrometry; phosphorylation; titanium dioxide.

FIGURE 1

Experimental workflow for automatic enrichment system. Steps in red involve injections from the autosampler, whereas those in blue correspond to length of time where mobile phase (MP) is allowed to flow through the column.

FIGURE 2

Loading study based on phenyl phosphate-binding capacity. (A) AUC intensity of all signals from phosphopeptides in black and nonphosphorylated peptides in gray. (B) Quantitative specificity of replicated enrichments plotted as a function of loading amount. (C) Number of unique spectra identified to phosphopeptides plotted in black and nonphosphorylated peptides plotted in gray.

FIGURE 3

Presence of phosphopeptides throughout each phase of enrichment. Venn overlap of unique spectra for all unique spectra (A) and for unique phosphopeptide spectra (B). (C) Summed AUC intensity for all phosphopeptide species (black) and nonphosphorylated peptides (gray) from fractions taken throughout the enrichment process performed at various enrichment flow rates.

FIGURE 4

Reproducibility of phosphopeptide enrichments at various flow rates. (A) Venn overlap of unique phosphopeptide spectra identified from two enrichments performed at 10 μL/min or 5 μL/min. (B) Log/log ratio plot of intensities of phosphopeptides in black and nonphosphorylated peptides in gray after LC-MS/MS analysis of enriched samples. Lines indicate a fold change of two. (C) Distribution of CV as a measure of quantitative reproducibility for each flow rate individually, and all samples combined regardless of enrichment flow rate.