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. 2011 Oct 15;83(20):7635-44.
doi: 10.1021/ac201894j. Epub 2011 Sep 20.

Rapid and Reproducible Single-Stage Phosphopeptide Enrichment of Complex Peptide Mixtures: Application to General and Phosphotyrosine-Specific Phosphoproteomics Experiments

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Rapid and Reproducible Single-Stage Phosphopeptide Enrichment of Complex Peptide Mixtures: Application to General and Phosphotyrosine-Specific Phosphoproteomics Experiments

Arminja N Kettenbach et al. Anal Chem. .
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Abstract

Reversible protein phosphorylation is an essential regulatory component of virtually every cellular process and is frequently dysregulated in cancer. However, significant analytical barriers persist that hamper the routine application of phosphoproteomics in translational settings. Here, we present a straightforward and reproducible approach for the broadscale analysis of protein phosphorylation that relies on a single phosphopeptide enrichment step using titanium dioxide microspheres from whole cell lysate digests and compared it to the well-established SCX-TiO(2) workflow for phosphopeptide purification on a proteome-wide scale. We demonstrate the scaleabilty of our approach from 200 μg to 5 mg of total NCI-H23 non-small cell lung adenocarcinoma cell lysate digest and determine its quantitative reproducibility by label-free analysis of phosphopeptide peak areas from replicate purifications (median CV: 20% RSD). Finally, we combine this approach with immunoaffinity phosphotyrosine enrichment, enabling the identification of 3168 unique nonredundant phosphotyrosine peptides in two LC-MS/MS runs from 8 mg of HeLa peptides, each with 80% phosphotyrosine selectivity, at a peptide FDR of 0.2%. Taken together, we establish and validate a robust approach for proteome-wide phosphorylation analysis in a variety of scenarios that is easy to implement in biomedical research and translational settings.

Figures

Figure 1
Figure 1. Parameter sweep for phosphopeptide purification using titanium dioxide (TiO2) and influence of sample complexity on phosphopeptide enrichment selectivity
(A.) Workflow of TiO2 enrichment. Cell lysate digests were separated on a SCX column, 24 fractions were collected and fraction #6–8 (acidic, early fractions, “EF”) and #18–20 (basic, late fractions, “LF”) were combined, enriched as described and analyzed by LC-MS/MS. (B.) Unique phosphopeptide identifications and selectivity purified at different TFA concentrations. (C.) Unique phosphopeptide identifications and selectivity purified at different lactic acid concentrations. (D.) Unique phosphopeptide identifications and selectivity purified with different amounts of TiO2. (E.) Unique phosphopeptide identifications and selectivity from incubations of variable time. (F.) Phosphopeptide selectivity when isolated from early, late, and combined (early + late) fractions the presence of 2M lactic acid in 50% ACN, 375μg TiO2 for 45 min. (G.) Phosphopeptide selectivity of peptides isolated from the combined fractions and plotted according to their origin from the early or late fractions.
Figure 2
Figure 2. Single-stage purification workflow for large-scale phosphoproteomics
(A.) Standard proteomics workflow for broadscale phosphorylation analysis. (B.) Single-stage proteomics workflow. Phosphopeptides are purified in a single, larger TiO2 enrichment step; the resulting phosphopeptides are separated by SCX chromatography and analyzed by LC-MS/MS. (C.) and (D.) Histograms depicting number of phosphopeptides (orange) and total peptides (blue) identified in each SCX fraction using the 24x-TiO2 (C.) and 1x-TiO2 (D.) approach. Phosphopeptide selectivity of each fraction is indicated (black). (E.) Total number of identified peptides, phosphopeptides, selectivity, and unique phosphorylation sites using the 24x-TiO2 and 1x-TiO2 approach. Overlap of unique phosphorylation sites (F.) and proteins (G.) identified using the 24x-TiO2 and 1x-TiO2 approaches.
Figure 3
Figure 3. Scalability of single-stage phosphopeptide purifications
(A.) Representative base peak chromatograms (fraction 8) of the SCX separation from 0.2mg, 1mg, and 5mg whole cell lysate single-stage purifications. (B.) Correlation coefficient of linear regression analyses of peptide peak area from 0.2mg to 5mg single-stage purifications. (C.) Slope of the least square lines of the linear regression analysis of peptide peak area from 0.2mg to 5mg single-stage purifications. (D.) Number of phosphopeptides (orange) and total peptides (blue) identified in the 0.2mg, 1mg and 5mg single-stage phosphopeptide enrichments. Phosphopeptide selectivity is indicated (black). (E.) Number of unique phosphorylation sites identified in the 0.2mg, 1mg and 5mg single-stage phosphopeptide enrichments. (F.) Overlap of unique phosphorylation sites identified in the 0.2mg, 1mg and 5mg single-stage phosphopeptide enrichments.
Figure 4
Figure 4. Reproducibility of single-stage phosphopeptide enrichments
(A.) Base peak chromatograms of replicate injections of one single-stage purification (sample A) from Jurkat lysate digest. (B.) Venn diagram depicting phosphopeptide occurrence in the four replicate injections of sample A. (C.) Histogram of peak area RSD of phosphopeptides from four replicate injections. (D.) Base peak chromatograms of replicate single-stage purifications (sample A, B. C, and D) from Jurkat lysate digest. (E.) Venn diagram depicting phosphopeptide occurrence in the four replicate TiO2 enrichments. (F.) Histogram of peak area RSD of phosphopeptides from four replicate TiO2 enrichments.
Figure 5
Figure 5. Phosphotyrosine enrichments from single-stage TiO2 purifications
(A). Workflow of phosphotyrosine enrichment from TiO2 enrichment. Phosphopeptides are purified in a single, large TiO2 enrichment step; from the resulting phosphopeptides, phosphotyrosine (pY) peptides are then isolated by immunoprecipitation (IP). (B.) Base peak chromatograms of the biological pY IP replicates. (C.) Significant motifs identified from the union of pY IP replicates. (D.) Insulin signaling pathway with pY sites identified in the replicate LC-MS/MS analyses indicated in red.

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