Methods apparatuses and systems for detecting and quantifying phosphoproteins
Embodiments herein provide methods, apparatuses, and systems for detecting, monitoring, measuring, and/or characterizing the activity of phosphoproteins such as tyrosine kinases (TKs) and downstream proteins in TK signal transduction pathways (e.g., TK pathway proteins). In various embodiments, the methods, apparatuses, and systems may use nanoparticles, such as quantum dots (QD), to detect and/or characterize the abnormally overactive TK signaling pathways that underlie tumorgenesis and tumor progression. In various embodiments, the QD-based methods, apparatuses, and systems may have a sufficiently high degree of sensitivity to enable the identification of new TK signaling pathway markers, for example for use in diagnosing, staging, monitoring, and/or prognosing cancers, or in evaluating the efficacy of cancer therapeutics.
1. A method of screening a drug for effectiveness in treating leukemia, the method comprising:
a) contacting a leukemia cell with the drug;
b) contacting the leukemia cell with a quantum dot labeled antibody specific for phosphorylated Abl, Akt, Crkl, ERK, STAT3, or STAT5;
c) localizing a set of quantum dots in a 3-dimensional quantum dot image stack of a sample using a computing device by the steps of:
i) receiving the 3-dimensional quantum dot image stack of the sample, wherein the sample includes a cell and the stack comprises a plurality of slices;
ii) generating a cumulative histogram of all pixel intensities greater than zero in the quantum dot image stack of the sample;
iii) calculating a low threshold and a high threshold based on the cumulative histogram;
iv) identifying a first pixel location of a candidate quantum dot in a first slice of the quantum dot image stack of the sample based on the low threshold and the high threshold;
v) pairing the first pixel location of the candidate quantum dot with a second pixel location of the candidate quantum dot in a second slice of the quantum dot image stack of the sample, wherein the second slice is adjacent to the first slice and pairing the first or second pixel location of the first candidate quantum dot with a third pixel location of the candidate quantum dot in a third slice of the quantum dot image stack of the sample wherein the third slice is adjacent to either the first slice or the second slice, thereby creating a quantum dot string for the candidate quantum dot;
vi) detecting a location, in the quantum dot image stack of the sample, of a membrane of the cell using a membrane staining image stack;
vii) determining the positions of the first and second quantum dots relative to the location of the membrane; and
viii) outputting a pixel location of the candidate quantum dot and a position of the candidate quantum dot relative to the membrane;
d) counting the number of quantum dots per cell in a set of the cells using fluorescence microscopy;
e) wherein a result showing that the average cell has less than 10 quantum dots per cell indicates that the drug is effective in treating leukemia.
2. The method of claim 1 wherein the leukemia cell is derived from a leukemia cell line.
3. The method of claim 2 wherein the cell line is the K562 cell line.
4. The method of claim 1 wherein the leukemia cell is derived from a sample from a subject.
5. The method of claim 1 wherein the drug is a known or putative tyrosine kinase inhibitor.
6. The method of claim 5 wherein the drug is dasatinib or a dasatinib homolog.
7. The method of claim 1 , wherein the membrane staining image stack is derived from a membrane-labeled cell.
8. The method of claim 7 wherein the membrane is labeled with wheat germ agglutinin.
9. The method of claim 1 wherein the low threshold is the intensity of the 95th percentile of the distribution of pixel intensity on the histogram +2(standard deviation of the distribution of pixel intensity) and the high threshold=the low threshold+the maximum pixel intensity/2.
10. The method of claim 9 wherein identifying the first pixel location of the candidate quantum dot comprises locating a first pixel that represents both a local maximum of intensity and that exceeds the high threshold.
11. The method of claim 10 further comprising identifying the first pixel location of the candidate quantum dot comprises locating a second pixel that exceeds the low threshold of intensity, exceeds the global intensity average, and is less than the high threshold of intensity.