Methods apparatuses and systems for detecting and quantifying phosphoproteins

Abstract

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 - 15 . (canceled) 
     
     
         16 . A method for quantitating protein activity in a biological sample comprising:
 labeling a protein in the biological sample with a label to form a labeled biological sample;   providing the labeled biological sample on a solid support; and automatically counting the labels, wherein automatically counting the labels includes:   automatically capturing an image of the labels in each of several Z-planes;   automatically detecting labels on the captured images; and   maintaining a count of discrete groups of labels or single labels.   
     
     
         17 . The method of  claim 16 , wherein the protein is a phosphoprotein. 
     
     
         18 . The method of  claim 16 , wherein the label comprises a nanoparticle probe with a detectable nanoparticle. 
     
     
         19 . The method of  claim 18 , wherein the detectable nanoparticle is capable of fluorescence. 
     
     
         20 . The method of  claim 16 , further comprising:
 positioning the labeled biological sample on a stage adapted to move the biological sample in X, Y, and Z planes;   automatically moving the labeled biological sample through multiple predetermined locations using the stage; and   capturing an image at each predetermined location to create a Z-stack.   
     
     
         21 . The method of  claim 20 , further comprising correcting for double counting of labels that appear in more than one image. 
     
     
         22 . The method of  claim 20 , further comprising binning the detected labels so that detected labels that are separated by less than a predetermined distance in any X, Y, or Z direction are each counted as a single label. 
     
     
         23 . The method of  claim 19 , wherein automatically detecting the nanoparticle probe comprises:
 applying an intensity threshold to a region encompassing the detected nanoparticle;   choosing a pixel within the region with a maximum intensity; and   determining a centroid of an intensity pattern around the chosen pixel.   
     
     
         24 . The method of  claim 18 , wherein the detectable nanoparticle comprises a semiconductor nanocrystal. 
     
     
         25 . The method of  claim 16 , wherein the protein is a tyrosine kinase or a tyrosine kinase pathway protein. 
     
     
         26 . The method of  claim 25 , wherein the label specifically binds to an active form of the tyrosine kinase pathway protein. 
     
     
         27 . The method of  claim 25 , wherein the tyrosine kinase or tyrosine kinase pathway protein is selected from the group of AATK, ABL1, ABL2; ALK, AXL, BLK, BMX, BTK, CSF1R, CSK, DDR1, DDR2, EGFR, EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHA10, EPHB1, EPHB2, EPHB3, EPHB4, EPHB6, ERBB2, ERBB3, ERBB4, FER, FES, FGFR1, FGFR2, FGFR3, FGFR4, FGR, FLT1, FLT3, FLT4, FRK, FYN, GSG2, HCK, IGF1R, ILK, INSR, INSRR, IRAK4, ITK, JAK1, JAK2, JAK3, KDR; KIT, KSR1, LCK, LMTK2, LMTK3, LTK, LYN, MATK, MERTK, MET, MLTK, MST1R, MUSK, NPR1, NTRK1, NTRK2, NTRK3, PDGFRA, PDGFRB, PLK4, PTK2, PTK2B, PTK6, PTK7, RET, ROR1, ROR2, ROS1, RYK, SGK493, SRC, SRMS, STYK1, SYK, TEC, TEK, TEX14, TIE1, TNK1, TNK2, TNNI3K, TXK, TYK2, TYRO3, YES1, and ZAP70 proteins. 
     
     
         28 . The method of  claim 25 , wherein the protein is selected from the group of a pAbI protein, a pAkt protein, a pCrkl protein, a pERK protein, a pSTAT3 protein, and a pSTAT5 protein. 
     
     
         29 . The method of  claim 16 , wherein the biological sample comprises a cancer cell. 
     
     
         30 . The method of  claim 29 , wherein the cancer cell is selected from the group of a non-small cell lung cancer cell, a chronic myeloid leukemia cell, and a gastrointestinal stromal tumor cell. 
     
     
         31 . The method of  claim 16 , wherein the biological sample comprises tumor tissue. 
     
     
         32 . The method of  claim 16 , wherein the biological sample comprises a formalin-fixed, paraffin-embedded tissue sample. 
     
     
         33 . The method of  claim 16 , wherein the biological sample is a single cell. 
     
     
         34 . The method of  claim 33 , wherein the single cell is a blood cell. 
     
     
         35 . The method of  claim 16 , wherein the biological sample comprises a stem cell. 
     
     
         36 . The method of  claim 16 , wherein the biological sample is a sample from a subject suspected of having cancer or known to have cancer. 
     
     
         37 . The method of  claim 16 , wherein the method is a method of diagnosing or prognosing cancer in a subject from which the biological sample is obtained and the diagnosing or prognosing is based at least upon the quantitated protein activity. 
     
     
         38 . The method of  claim 18 , wherein the nanoparticle probe comprises an antibody. 
     
     
         39 . The method of  claim 38 , wherein the antibody is selected from the group of an anti-Abl antibody, an anti-Akt antibody, an anti-Crkl antibody, an anti-ERK antibody, an anti-STAT3 antibody, an anti-STAT5 antibody, an anti-pAbl antibody, an anti-pAkt antibody, an anti-pCrkl antibody, an anti-pERK antibody, an anti-pSTAT3 antibody, and an anti-pSTAT5 antibody. 
     
     
         40 . The method of  claim 16 , wherein the method is a method of determining drug effectiveness based at least upon the quantitated protein activity.