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Age-related macular degeneration (AMD) describes the progressive degeneration of the retinal pigment epithelium (RPE), retina, and choriocapillaris and is the leading cause of blindness in people over 50. The molecular mechanisms underlying this multifactorial disease remain largely unknown. To uncover novel secretory biomarkers related to the pathogenesis of AMD, we adopted an integrated approach to compare the proteins identified in the conditioned medium (CM) of cultured RPE cells and the exosomes derived from CM and from the aqueous humor (AH) of AMD patients by LC-ESI-MS/MS. Finally, LC-MRM was performed on the AH from patients and controls, which revealed that cathepsin D, cytokeratin 8, and four other proteins increased in the AH of AMD patients. The present study has identified potential biomarkers and therapeutic targets for AMD treatment, such as proteins related to the autophagy-lysosomal pathway and epithelial-mesenchymal transition, and demonstrated a novel and effective approach to identifying AMD-associated proteins that might be secreted by RPE in vivo in the form of exosomes. The proteomics-based characterization of this multifactorial disease could help to match a particular marker to particular target-based therapy in AMD patients with various phenotypes.

CtBP is a transcriptional repressor which plays a significant role in the regulation of cell proliferation and tumor progression. It was reported that glucose withdrawal causes induction of Bax due to the dissociation of CtBP from the Bax promoter. However, the precise mechanism involved in the regulation of CtBP still remains unclear. In this study, we found that an activated AMP-activated protein kinase (AMPK) phosphorylates CtBP1 on Ser-158 upon metabolic stresses. Moreover, AMPK-mediated phosphorylation of CtBP1 (S158) attenuates the repressive function of CtBP1. We also confirmed that triggering activation of AMPK by various factors resulted in an increase of Bax gene expression. These findings provide connections of AMPK with CtBP1-mediated regulation of Bax expression for cell death under metabolic stresses.

Microvesicles (MV) are membrane vesicles secreted from the plasma and endosomal membrane compartment by various cell types such as hematopoietic, epithelial, and tumor cells. Actively growing tumor cells shed MV, and the rate of shedding increases in malignant tumors. Although recent progress in this area has revealed that tumor-derived MV play multiple roles in tumor growth and metastasis via immune escape, tumor invasion, and angiogenesis, the mechanism of vesicle formation and the biological roles of tumor-derived MV are not understood. Here, we report the first global proteomic analysis of highly purified MV from human colorectal cancer cells. Using 1D SDS gel electrophoresis and nano-LC-MS/MS analyses, we identified a total of 547 microvesicular proteins from three independent experiments with high confidence; 416 proteins were identified at least in two trials, including 181 as yet unreported proteins. We identified 49 proteins involved in the biogenesis of MV, including annexins, ADP-ribosylation factors, and Rab proteins. We also identified 28 proteins that may function in tumorigenesis via promotion of migration, invasion, and growth of tumor cells, immune modulation, metastasis, and angiogenesis. Taken together with previously reported results, our observations suggest that tumor-derived MV may act as communicasomes, that is, extracellular organelles that play diverse roles in intercellular communication. This information will help elucidate the biogenesis and functions of tumor-derived MV, and aid in the development of effective vaccines for various cancers, including colorectal cancer.

Gram-negative bacteria constitutively secrete native outer membrane vesicles (OMVs) into the extracellular milieu. Although recent progress in this area has revealed that OMVs are essential for bacterial survival and pathogenesis, the mechanism of vesicle formation and the biological roles of OMVs have not been clearly defined. Using a proteomics approach, we identified 141 protein components of Escherichia coli-derived native OMVs with high confidence; two separate analyses yielded identifications of 104 and 117 proteins, respectively, with 80 proteins overlapping between the two trials. In the group of identified proteins, the outer membrane proteins were highly enriched, whereas inner membrane proteins were lacking, suggesting that a specific sorting mechanism for vesicular proteins exists. We also identified proteins involved in vesicle formation, the removal of toxic compounds and attacking phage, and the elimination of competing organisms, as well as those involved in facilitating the transfer of genetic material and protein to other bacteria, targeting host cells, and modulating host immune responses. This study provides a global view of native bacterial OMVs. This information will help us not only to elucidate the biogenesis and functions of OMV from nonpathogenic and pathogenic bacteria but also to develop vaccines and antibiotics effective against pathogenic strains.

Peptide nucleic acid (PNA) probes were designed to bind to the internal reference sequence and the single base mutation sequence within PCR-amplified DNA templates. PNAs hybridized to the target sequences on DNA were analyzed using MALDI-TOF mass spectrometry. Accurate quantification of the relative amount of mutant DNA was reproducibly demonstrated.

Noncoding RNAs have drawn significant attention in biology recently. Whereas the current research is highly inclined to microRNAs, research on other noncoding RNAs has lagged behind. Here, we investigated a novel noncoding RNA that has been known as precursor microRNA miR-886 (pre-miR-886). Pre-miR-886 has been proposed also as a vault RNA, a component of the vault complex implicated in cancer drug resistance. We identified pre-miR-886 as a 102-nucleotide-long, abundant cytoplasmic RNA that is neither a genuine pre-microRNA nor a vault RNA. Pre-miR-886 is physically associated with PKR (Protein Kinase RNA-activated), an interferon-inducible and double-stranded RNA dependent kinase. The suppression of pre-miR-886 activates PKR and its downstream pathways, eIF2α phosphorylation and the NF-κB pathway, leading to impaired cell proliferation. We also found that pre-miR-886 is suppressed in a wide-range of cancer cell lines and in clinical specimens. This study is the first intense characterization of pre-miR-886 as well as the initial report on its function as a PKR regulator, which suggests a critical role in tumorigenesis.

The goal of this study was to identify promising new biomarkers of cadmium by identifying differentially expressed proteins in Paronychiurus kimi after exposure to cadmium. Through proteomic analysis of P. kimi using 1-D PAGE and nano-LC-MS/MS, 36 downregulated proteins and 40 upregulated proteins were found. Some of the downregulated and upregulated proteins were verified by LC-MS/MS analysis after 2-D PAGE. Downregulated proteins in response to cadmium exposure were involved in glycolysis and energy metabolism, chaperones, transcription, reproduction, and neuron growth. In contrast, proteins involved in glycolysis and energy production, neurogenesis, defense systems response to bacteria, and protein biosynthesis were upregulated in cadmium-treated collembolans. Cubulin may be a potential biomarker for the detection of cadmium in P. kimi since this biomarker was able to low levels (3.5 mg/kg) of cadmium. The 14-3-3 ζ was also found to be a potential biomarker for the detection of medium levels (14 mg/kg) of cadmium. Collembolans may be an alternative tool to humans because many collembolans proteins show a high homology to human proteins.

Direct tissue imaging mass spectrometry (IMS) by matrix-assisted laser desorption ionization and time-of-flight (MALDI-TOF) mass spectrometry has become increasingly important in biology and medicine, because this technology can detect the relative abundance and spatial distribution of interesting proteins in tissues. Five thyroid cancer samples, along with normal tissue, were sliced and transferred onto conductive glass slides. After laser scanning by MALDI-TOF equipped with a smart beam laser, images were created for individual masses and proteins were classified at 200-µm spatial resolution. Based on the spatial distribution, region-specific proteins on a tumor lesion could be identified by protein extraction from tumor tissue and analysis using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Using all the spectral data at each spot, various intensities of a specific peak were detected in the tumor and normal regions of the thyroid. Differences in the molecular weights of expressed proteins between tumor and normal regions were analyzed using unsupervised and supervised clustering. To verify the presence of discovered proteins through IMS, we identified ribosomal protein P2, which is specific for cancer. We have demonstrated the feasibility of IMS as a useful tool for the analysis of tissue sections, and identified the tumor-specific protein ribosomal protein P2. Graphical Abstract Keywords: Pathology, Proteins, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Thyroid Gland, Neoplasms INTRODUCTION Papillary thyroid carcinomas (PTC) are the most common form of thyroid cancer, accounting for nearly 85% of primary thyroid malignancies. Commonly, the solitary nodule is a palpably discrete swelling within an otherwise apparently normal thyroid gland, and is incidentally detected by radiological evaluation (1). From a clinical standpoint, the possibility of neoplastic disease is a major cause for concern in patients who present with thyroid nodules, because most conventional papillary carcinomas present as asymptomatic thyroid nodules. Recent studies have demonstrated 2 molecular mechanisms that function in the carcinogenesis of thyroid cancer: the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K)/Akt pathways (2, 3). Additionally, such pathways are related to genetic alterations such as rearrangements in the "rearranged during transfection" (RET) or neurotrophic tyrosine kinase receptor 1 (NTRK1) genes, and the activation of point mutations in the BRAF gene (4). In a previous study, protein expression patterns according to genetic variables have been demonstrated to correlate with the microscopic features, clinical manifestations, and prognostic characteristics of PTC (5). Although various protein alterations have been detected as diagnostic markers or prognostic predictors, the challenge remains to identify the expression of new molecules in tumors. In biological research, protein expression profiling technology is a useful method to identify differential protein expression patterns and modifications. Analytical techniques with high sensitivity and increased throughput are required for the discovery of new biomarkers and new drugs. Recent progress in imaging mass spectrometry (IMS) has made it possible to identify several cellular components such as proteins, drugs, and other endogenous molecules directly on tissue sections (6, 7, 8, 9, 10). IMS uses matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) technology. The matrix is applied on cryosectioned tissue and a very small area of the matrix-applied tissue is analyzed using MALDI-MS. Differences between the analyzed areas are displayed by the imaging program. Each analyzed area of the tissue section, which can be as small as >200 µm, on a conductive surface such as gold-plated or indium tin oxide (ITO)-coated slide, is analyzed spot by spot using MALDI-MS after the application of the matrix. Using all the spectral data from each spot, the magnitudes of specific peaks on each spot can be displayed in terms of color intensity. In this way, spatial information on the tissue can be obtained. Based on the spatial distribution, region-specific proteins on a tissue can be identified by extracting proteins from the tissue, digesting them with trypsin, and analyzing the fragments using liquid chromatography with tandem MS (LC-MS/MS). This technology using IMS has broad applications in the detection of new proteins in various fields. Compared with conventional imaging methods, the advantages of IMS are that it does not require the additional use of a specific antibody against the protein, and that it integrates histopathology and protein expression (11). For this reason, IMS provides superior information regarding distinct molecular arrangements in tissue sections. In the present study, we attempted to analyze thyroid samples, including those from PTC, using IMS. Before analysis using IMS, the diagnosis was confirmed by microscopic evaluation, immunohistochemical (IHC) staining of markers such as CK19, galectin-3, and RET, and pyrosequencing of the BRAF mutation, and then 5 homogenous samples of PTC were selected. After comparison of the differential molecular weight distribution between normal and tumor tissues using IMS, a protein of a specific molecular weight was identified by sequencing combined with LC-MS/MS, and then the presence of this protein was reconfirmed by western blot analysis.