Protein analysis using solid-state nanopores is challenging due to limitations in bandwidth and signal-to-noise ratio. Recent improvements of those two aspects have made feasible the study of small peptides using solid-state nanopores, which have an advantage over biological counterparts in tunability of the pore diameter. Here, we report on the detection and characterization of peptides as small as 33 amino acids. Silicon nitride nanopores with thicknesses less than 10 nm are used to provide signal-to-noise (S/N) levels up to S/N ∼ 10 at 100 kHz. We demonstrate differentiation of monomer and dimer forms of the GCN4-p1 leucine zipper, a coiled-coil structure well studied in molecular biology, and compare with the unstructured 33-residue monomer. GCN4-p1 is sequence segment associated with homodimerization of the transcription factor General Control Nonderepressible 4 (GCN4), which is involved in the control of amino acid synthesis in yeast. The differentiation between two oligomeric forms demonstrates the capabilities of improved solid-state nanopore platforms to extract structural information involving short peptide structures.

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Glioblastoma multiforme (GBM), the most aggressive and most common malignant adult brain tumor, accounts for 50% of all gliomas and it is classified histologically as grade IV. Growing evidence for intratumor heterogeneity shows that single-site biopsies or selected profiling of a portion of a resected tumor falls short of revealing the complete genomic landscape of the entire tumor. EGFR amplification occurs in 50% of primary GBM tumors, and is frequently associated with the expression of a constitutively active, ligand-independent mutant form of the receptor, EGFRvIII. Glioma circulating tumor cells (gCTCs) have recently been identified in the blood of glioma patients by our research group. In this study, we report that gCTCs can be captured, enumerated and genetically characterized for EGFRvIII. To achieve this goal, we have thus far a) detected gCTCs through a Telomerase promoterbased assay, b) isolated individual gCTCs using a vacuum-assisted capillary microdissection approach, c) performed whole transcriptome amplification and d) analyzed the expression of EGFRviii by qPCR under optimized conditions. Furthermore, our group is conducting an IRB-approved pilot study with the purpose of examining the feasibility of this approach (to support initiation of an expanded study). In this study, we seek to characterize the genetic heterogeneity of individual gCTCs from high grade glioma patients. This approach would have major impact as it will clarify the biologically-relevant question of heterogeneity of gCTCs in a given patient with glioma, predict whether a patient would respond or be resistant to a molecularly targeted therapy, unravel the overall mechanistic underpinnings of a given patient's brain tumor, and lastly, track the biological evolution of the tumor over the patient's treatment course and post-treatment period. Our ultimate goal is to streamline predictive testing and effectively utilize a Neurooncology targeted biomarker to benefit patients.