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. 2014 Mar;28(3):716-9.
doi: 10.1038/leu.2013.347. Epub 2013 Nov 19.

Global phosphoproteome analysis of human bone marrow reveals predictive phosphorylation markers for the treatment of acute myeloid leukemia with quizartinib

C Schaab  1 , F S Oppermann  2 , M Klammer  2 , H Pfeifer  3 , A Tebbe  2 , T Oellerich  4 , J Krauter  5 , M Levis  6 , A E Perl  7 , H Daub  2 , B Steffen  3 , K Godl  2 , H Serve  4
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Free PMC article

Global phosphoproteome analysis of human bone marrow reveals predictive phosphorylation markers for the treatment of acute myeloid leukemia with quizartinib

C Schaab et al. Leukemia. 2014 Mar.
Free PMC article
No abstract available

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Figures

Figure 1
Figure 1
Workflow of processing bone marrow aspirates and global quantitative phosphoproteome analysis. The leukemia cells were isolated using density-gradient centrifugation and stored as vital cells for further processing at −80 °C. Equal amounts of lysates from blasts and Super-SILAC-standard were mixed. Proteins were extracted and digested with trypsin. The resulting peptides were separated into 12 fractions by strong cation exchange (SCX) chromatography and the phosphopeptides were enriched using immobilized metal affinity chromatography (IMAC). High-resolution LC-MS/MS data were processed using the MaxQuant software. Data from 12 patients (six responders and six non-responders) were used in the classification workflow for selection of five predictive phosphorylation sites (phospho-signature) and for training of a support vector machine. Classification accuracy was estimated with leave-one-out cross-validation. Finally, the signature was applied to nine independent validation samples.
Figure 2
Figure 2
Identification of predictive phospho-signature. (a) Scatter plot showing the mean log-ratios (AML sample vs spike-in SILAC reference) for the responder (y axis) and non-responder (x axis) samples. Each dot represents one phosphorylation site. The three significantly differential sites are marked red. (b) Cross-validation results represented by the probability for assignment to the responder class. Responders (left half) are predicted correctly if they are assigned a probability >0.5; non-responder (right half) are correct if they are assigned a probability <0.5. (c) Heat map of the final five selected phosphorylation sites. Rows are the 12 training sample, columns are the phospho-sites ordered by their importance ranks (left is the best). Red indicates up-, blue downregulation, gray no regulation. Missing values are colored white. (d) Validation results of nine independent patients. The true and predicted responses are compared.
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References

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