Leukemia relapse occurs in at least 20% of children with acute lymphoblastic leukemia treated with chemotherapy drugs. This observation leads to the hypothesis that the proteomes of these two groups, distinguished by the occurrence of relapse, differ in their specific protein expression patterns and protein post-translational modifications. Protein-based biomarkers from minimal residual disease following treatment of acute lymphoblastic leukemia using chemotherapy drugs have great potential as new diagnostic tool. The levels of up- and down-modification of peptides and proteins resulting from diseased cell may signal various clinical features and diagnostic clues. We present a method that uses zirconium phosphate beads to purify phosphopeptides and phosphoprotein, followed with SELDI mass spectrometry to study phosphoproteomic patterns.
The cell growth process in response to treatment with the chemotherapy drug doxorubicin was assessed with a hemacytometer over a three day period. The result is shown in Figure 1. When REH human leukemia cells were treated with 8 µL of 290 nM doxorubicin, the cell concentration in the media was nearly constant at 0.5 million cells per milliliter. For the control sample which was not treated with doxorubicin, the cell concentration tripled in the same time period.
Fig. 1. Cell growth process in response to treatment with (red dot) and without (black dot) the chemotherapy drug doxorubicin
The phosphopeptide profile of the leukemia cell extract following treatment was analyzed daily for three days using SELDI MS and compared to the untreated extract. We detected 15 major peaks in the mass spectrum from the isolated phosphopeptides from the doxorubicin treated cells that monotonically decreased: m/z 1916, 2115, 2126, 2165, 2193, 2214, 2242, 2320, 2516, 2723, 3558, 3481, 4290, 4410, and 4610. We also detected 3 peaks in the mass spectrum from the isolated phosphopeptides specific to the doxorubicin treated cells that monotonically increased: m/z 4580, 6210, and 6290. These 18 peaks comprise a phosphopeptide profile for the early diagnosis of minimal residual disease in response to doxorubicin treatment of leukemia cell disease.