A Robust Error Model for iTRAQ Quantification Reveals Divergent Signaling between Oncogenic FLT3 Mutants in Acute Myeloid Leukemia

Abstract

The FLT3 receptor tyrosine kinase plays an important role in normal hematopoietic development and leukemogenesis. Point mutations within the activation loop and inframe tandem duplications of the juxtamembrane domain represent the most frequent molecular abnormalities observed in acute myeloid leukemia. Interestingly these gain-of-function mutations correlate with different clinical outcomes, suggesting that signals from constitutive FLT3 mutants activate different downstream targets. In principle, mass spectrometry offers a powerful means to quantify protein phosphorylation and identify signaling events associated with constitutively active kinases or other oncogenic events. However, regulation of individual phosphorylation sites presents a challenging case for proteomics studies whereby quantification is based on individual peptides rather than an average across different peptides derived from the same protein. Here we describe a robust experimental framework and associated error model for iTRAQ-based quantification on an Orbitrap mass spectrometer that relates variance of peptide ratios to mass spectral peak height and provides for assignment of p value, q value, and confidence interval to every peptide identification, all based on routine measurements, obviating the need for detailed characterization of individual ion peaks. Moreover, we demonstrate that our model is stable over time and can be applied in a manner directly analogous to ubiquitously used external mass calibration routines. Application of our error model to quantitative proteomics data for FLT3 signaling provides evidence that phosphorylation of tyrosine phosphatase SHP1 abrogates the transformative potential, but not overall kinase activity, of FLT3-D835Y in acute myeloid leukemia. Molecular & Cellular Proteomics 9:780 -790, 2010.Deregulated tyrosine phosphorylation is a well established molecular hallmark in the development of both solid tumors and hematopoietic malignancies (1, 2). In acute myeloid leukemia (AML), 1 mutations of the Fms-like tyrosine kinase 3 (Flt3) gene or its overexpression represent the most frequent molecular abnormalities, observed in ϳ35 and ϳ90% of patients, respectively (3, 4). Internal tandem duplication (ITD) in the juxtamembrane region occurs in ϳ25% of patients, whereas another 7% of cases manifest as point mutations in the activation loop at Asp-835 (typically D835Y). Both mutation classes induce constitutive tyrosine kinase activity and confer IL-3-independent growth of the factor IL-3-dependent BaF3 and 32D cells. Interestingly, the ITD and D835Y mutants respond differently to the current suite of targeted therapeutics (5), and in vitro studies suggest that they can activate different downstream targets, including STAT5a (6 -8). Collectively, these observations motivate continued efforts to delineate oncogenic pathways in further detail to expand our understanding of the molecular basis for disease progression and to identify additional therapeutic entry points.Over the past decade, mass spectrometry-based pr...

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