From aptamer-based biomarker discovery to diagnostic and clinical applications: an aptamer-based, streamlined multiplex proteomic assay

Recently, we reported an aptamer-based, highly multiplexed assay for the purpose of biomarker identification. To enable seamless transition from highly multiplexed biomarker discovery assays to a format suitable and convenient for diagnostic and life-science applications, we developed a streamlined, plate-based version of the assay. The plate-based version of the assay is robust, sensitive (sub-picomolar), rapid, can be highly multiplexed (upwards of 60 analytes), and fully automated. We demonstrate that quantification by microarray-based hybridization, Luminex bead-based methods, and qPCR are each compatible with our platform, further expanding the breadth of proteomic applications for a wide user community

From SOMAmer-Based Biomarker Discovery to Diagnostic and Clinical Applications: A SOMAmer-Based, Streamlined Multiplex Proteomic Assay

Recently, we reported a SOMAmer-based, highly multiplexed assay for the purpose of biomarker identification. To enable seamless transition from highly multiplexed biomarker discovery assays to a format suitable and convenient for diagnostic and life-science applications, we developed a streamlined, plate-based version of the assay. The plate-based version of the assay is robust, sensitive (sub-picomolar), rapid, can be highly multiplexed (upwards of 60 analytes), and fully automated. We demonstrate that quantification by microarray-based hybridization, Luminex bead-based methods, and qPCR are each compatible with our platform, further expanding the breadth of proteomic applications for a wide user community

Aptamer-based multiplexed proteomic technology for biomarker discovery

Interrogation of the human proteome in a highly multiplexed and efficient manner remains a coveted and challenging goal in biology. We present a new aptamer-based proteomic technology for biomarker discovery capable of simultaneously measuring thousands of proteins from small sample volumes (15 [mu]L of serum or plasma). Our current assay allows us to measure ~800 proteins with very low limits of detection (1 pM average), 7 logs of overall dynamic range, and 5% average coefficient of variation. This technology is enabled by a new generation of aptamers that contain chemically modified nucleotides, which greatly expand the physicochemical diversity of the large randomized nucleic acid libraries from which the aptamers are selected. Proteins in complex matrices such as plasma are measured with a process that transforms a signature of protein concentrations into a corresponding DNA aptamer concentration signature, which is then quantified with a DNA microarray. In essence, our assay takes advantage of the dual nature of aptamers as both folded binding entities with defined shapes and unique sequences recognizable by specific hybridization probes. To demonstrate the utility of our proteomics biomarker discovery technology, we applied it to a clinical study of chronic kidney disease (CKD). We identified two well known CKD biomarkers as well as an additional 58 potential CKD biomarkers. These results demonstrate the potential utility of our technology to discover unique protein signatures characteristic of various disease states. More generally, we describe a versatile and powerful tool that allows large-scale comparison of proteome profiles among discrete populations. This unbiased and highly multiplexed search engine will enable the discovery of novel biomarkers in a manner that is unencumbered by our incomplete knowledge of biology, thereby helping to advance the next generation of evidence-based medicine

Protein Signature of Lung Cancer Tissues

Lung cancer remains the most common cause of cancer-related mortality. We applied a highly multiplexed proteomic technology (SOMAscan) to compare protein expression signatures of non small-cell lung cancer (NSCLC) tissues with healthy adjacent and distant tissues from surgical resections. In this first report of SOMAscan applied to tissues, we highlight 36 proteins that exhibit the largest expression differences between matched tumor and non-tumor tissues. The concentrations of twenty proteins increased and sixteen decreased in tumor tissue, thirteen of which are novel for NSCLC. NSCLC tissue biomarkers identified here overlap with a core set identified in a large serum-based NSCLC study with SOMAscan. We show that large-scale comparative analysis of protein expression can be used to develop novel histochemical probes. As expected, relative differences in protein expression are greater in tissues than in serum. The combined results from tissue and serum present the most extensive view to date of the complex changes in NSCLC protein expression and provide important implications for diagnosis and treatment

Single Ribosomal Protein Mutations in Antibiotic-Resistant Bacteria Analyzed by Mass Spectrometry

Mutations in several ribosomal proteins are known to be related to antibiotic resistance. For several strains of Escherichia coli, the mutated protein is known but the amino acid actually altered has not been documented. Characterization of these determinants for antibiotic resistance in proteins will further the understanding of the precise mechanism of the antibiotic action as well as provide markers for resistance. Mass spectrometry can be used as a valuable tool to rapidly locate and characterize mutant proteins by using a small amount of material. We have used electrospray and matrix-assisted laser desorption ionization–time of flight (MALDI–TOF) mass spectrometry to map out all 56 ribosomal proteins in E. coli based on intact molecular masses. We used this fingerprinting approach to locate variants of ribosomal proteins displaying a change in mass. In particular we have studied proteins responsible for streptomycin, erythromycin, and spectinomycin resistance in three strains of E. coli, and then we characterized each mutation responsible for resistance by analyzing tryptic peptides of these proteins by using MALDI-TOF and nanoelectrospray tandem mass spectrometry. The results provided markers for antibiotic resistance and demonstrated that mass spectrometry can be used to rapidly investigate changes in individual proteins from a complex with picomole amounts of protein

Patient demographics, resection location and tumor types for the eight NSCLC analyzed samples.

<p>Patient demographics, resection location and tumor types for the eight NSCLC analyzed samples.</p

List of potential NSCLC biomarkers identified from serum and tissue samples.

<p>Proteins identified in both studies are shown in boldface font.</p

Thrombospondin-2 (TSP-2) histochemical identification in tissue samples.

<p>TSP-2 is identified in serial frozen sections of a single lung carcinoma specimen by (A) a home-made rabbit polyclonal TSP-2 polyclonal antibody, (B) the pre-immune serum from rabbits used to make the home-made polyclonal antibody, (C) a commercial (Novus) rabbit polyclonal TSP-2 antibody, and (D) the TSP-2 SOMAmer. The TSP-2 SOMAmer was used to stain frozen sections of normal and malignant lung tissue, with standard Avidin-Biotin-Peroxidase color development, to demonstrate different morphologic distributions: (E) Strong staining of the fibrotic stroma surrounding tumor nests, with minimal cytosolic staining of carcinoma cells, (F) Strong staining of the fibrotic stroma surrounding tumor nests in a mucinous adenocarcinoma, with no significant staining of the carcinoma cells, (G) normal lung tissue, showing strong cytosolic staining of bronchial epithelium and scattered alveolar macrophages, and (H) strong cytosolic staining of an adenocarcinoma, with no significant staining of the non-fibrotic, predominantly inflammatory stroma.</p