Methods and compositions for targeted two-dimensional western blot analysis for early cancer detection and cancer diagnosis up to ten years in advance of clinical symptoms of malignant disease

A calibrated method of quantitation of ENOX2 transcript variants on a western blot comprising:

• subjecting a sample from the subject concentrated sample to electrophoretic protein separation;
• western blotting the electrophoretically separated proteins onto a protein capture film or paper;
• activating one or more ENOX2 transcript variants on the protein capture film or paper with a reduced ENOX2 substrate;
• and
• detecting the presence of the one or more activated ENOX2 transcript variants using a pan-ENOX2 detectable binding reagent, wherein a 10 to 100 fold increase in detection sensitivity for ENOX2 is obtained when compared to the non-activated ENOX2, wherein the amount of one or more activated ENOX2 transcript variants is calibrated based on a spot diameter and an intensity of the ENOX2 transcript variant on the protein capture film or paper when compared to a known amount of the one or more ENOX2 transcript variants.

A method for determining the tissue of origin of a cancer in a subject comprising:

• subjecting proteins in a sample from the subject to a two-dimensional (2D) molecular weight and isoelectric point electrophoretic protein separation;
• transferring the proteins to a protein capture film or paper;
• activating the electrophoretically separated ENOX2 transcript variants with an ENOX2 electron donor on the protein capture film or paper;
• detecting the activated ENOX2 transcript variants;
• and
• determining the tissue of origin of the cancer based on the presence of one or more activated ENOX2 transcript variants selected from Bladder, Blood Cell, Breast, Cervical, Colorectal, Esophageal, Gastric, Hepatocellular, Leukemias, Lung Non-Small Cell, Lung Small Cell, Lymphomas, Melanoma, Mesothelioma, Myeloma, Ovarian, Pancreatic, Prostate, Renal Cell (Kidney), Sarcoma, Squamous Cell, Testicular Germ Cell, Thyroid Follicular, or Thyroid Papillary, wherein activation of the one or more ENOX2 transcript variants in situ increases the detection sensitivity by 10 to 100 fold when compared to non-activated ENOX2 transcript variants.