Combined blood/tissue analysis for cancer biomarker discovery: application to renal cell carcinoma

Abstract

A method that relies on subtractive tissue-directed shot-gun proteomics to identify tumor proteins in the blood of a patient newly diagnosed with cancer is described. To avoid analytical and statistical biases caused by physiologic variability of protein expression in the human population, this method was applied on clinical specimens obtained from a single patient diagnosed with nonmetastatic renal cell carcinoma (RCC). The proteomes extracted from tumor, normal adjacent tissue and preoperative plasma were analyzed using 2D-liquid chromatography-mass spectrometry (LC-MS). The lists of identified proteins were filtered to discover proteins that (i) were found in the tumor but not normal tissue, (ii) were identified in matching plasma, and (iii) whose spectral count was higher in tumor tissue than plasma. These filtering criteria resulted in identification of eight tumor proteins in the blood. Subsequent Western-blot analysis confirmed the presence of cadherin-5, cadherin-11, DEAD-box protein-23, and pyruvate kinase in the blood of the patient in the study as well as in the blood of four other patients diagnosed with RCC. These results demonstrate the utility of a combined blood/tissue analysis strategy that permits the detection of tumor proteins in the blood of a patient diagnosed with RCC.

Figure 1

a – g. The outline of methodology for identification of tumor proteins in peripheral blood of a patient diagnosed with non-metastatic RCC.

Figure 2

a – d. Venn diagrams depicting subtractive proteomic analysis employed to reveal identities of tumor-residing proteins. A total of 202 proteins were identified by at least two protein specific peptides in any of the peptide fractions from tumorous tissue but not in any of the technical replicates from normal adjacent tissue (kidney). Subsequent analysis revealed identities of 8 tumor-residing proteins in the plasma by comparing/subtracting proteins identified exclusively in the tumor (202) and those identified in plasma (179). These proteins exhibited higher total peptide count in tumor vs. plasma and are considered as genuine tumor proteins (a). Secondary structure of cadherin-5 depicting the location of identified peptides. Peptides in red font were identified in tumor while peptides depicted by blue font were identified in plasma. All identified peptides in tumor and plasma reside in extracellular domain of this integral plasma membrane protein (b). Extracted ion chromatograms of the KPLIGTVLAMDPDAAR peptide identifying cadherin-5 in tumor (red font) and peripheral plasma (blue font) indicating higher concentration level of this peptide/protein in tumor (c). Western blot analysis of cadherin-5. A total of 20 μg of depleted plasma protein from the patient under study (2) and a total of 20 μg depleted plasma protein from matched healthy donor (3) along with 30 μg of depleted plasma protein from the same patient (5) and 30 μg depleted plasma protein from matched healthy donor (6) were separated on 4-20% Tris-Glycine gradient gels. Also, a total of 20 μg of cellular lysates: HUVEC (7), LNCaP (8) and SKOV3 (9) were separated using the same gradient gel and transferred to Immuno-Blot PVDF membranes. The membranes were blocked by 3% bovine serum albumin and then probed overnight at 4 °C with anti-cadherin-5 MAb followed by peroxidase conjugated goat anti-mouse IgG secondary antibody (d).