Exosomes are increasingly recognized as important mediators of cell-cell communication in cancer progression through the horizontal transfer of RNAs and proteins to neighboring or distant cells. Hepatocellular carcinoma (HCC) is a highly malignant cancer, whose metastasis is largely influenced by the tumor microenvironment. The possible role of exosomes in the interactions between HCC tumor cell and its surrounding hepatic milieu are however largely unknown. In this study, we comprehensively characterized the exosomal RNA and proteome contents derived from three HCC cell lines (HKCI-C3, HKCI-8 and MHCC97L) and an immortalized hepatocyte line (MIHA) using Ion Torrent sequencing and mass spectrometry, respectively. RNA deep sequencing and proteomic analysis revealed exosomes derived from metastatic HCC cell lines carried a large number of protumorigenic RNAs and proteins, such as MET protooncogene, S100 family members and the caveolins. Of interest, we found that exosomes from motile HCC cell lines could significantly enhance the migratory and invasive abilities of non-motile MIHA cell. We further demonstrated that uptake of these shuttled molecules could trigger PI3K/AKT and MAPK signaling pathways in MIHA with increased secretion of active MMP-2 and MMP-9. Our study showed for the first time that HCC-derived exosomes could mobilize normal hepatocyte, which may have implication in facilitating the protrusive activity of HCC cells through liver parenchyma during the process of metastasis.

Preterm infants are highly susceptible to life-threatening infections that are clinically difficult to detect, such as late-onset septicemia and necrotizing enterocolitis (NEC). Here, we used a proteomic approach to identify biomarkers for diagnosis of these devastating conditions. In a case-control study comprising 77 sepsis/NEC and 77 nonsepsis cases (10 in each group being monitored longitudinally), plasma samples collected at clinical presentation were assessed in the biomarker discovery and independent validation phases. We validated the discovered biomarkers in a prospective cohort study with 104 consecutively suspected sepsis/NEC episodes. Proapolipoprotein CII (Pro-apoC2) and a des-arginine variant of serum amyloid A (SAA) were identified as the most promising biomarkers. The ApoSAA score computed from plasma apoC2 and SAA concentrations was effective in identifying sepsis/NEC cases in the case-control and cohort studies. Stratification of infants into different risk categories by the ApoSAA score enabled neonatologists to withhold treatment in 45% and enact early stoppage of antibiotics in 16% of nonsepsis infants. The negative predictive value of this antibiotic policy was 100%. The ApoSAA score could potentially allow early and accurate diagnosis of sepsis/NEC. Upon confirmation by further multicenter trials, the score would facilitate rational prescription of antibiotics and target infants who require urgent treatment.

Background: Detection of hepatocellular carcinoma (HCC) in patients with chronic liver disease (CLD) is difficult. We investigated the use of comprehensive proteomic profiling of sera to differentiate HCC from CLD. Methods: Proteomes in sera from 20 CLD patients with ␣-fetoprotein (AFP) <500 g/L (control group) and 38 HCC patients (disease group) were profiled by anionexchange fractionation (first dimension), two types (IMAC3 copper and WCX2) of ProteinChip ® Arrays (second dimension), and time-of-flight mass spectrometry (third dimension). Bioinformatic tests were used to identify tumor-specific proteomic features and to estimate the values of the tumor-specific proteomic features in the diagnosis of HCC. Cross-validation was performed, and we also validated the models with pooled sera from the control and disease groups, serum from a CLD patient with AFP >500 g/L, and postoperative sera from two HCC patients. Results: Among 2384 common serum proteomic features, 250 were significantly different between the HCC and CLD cases. Two-way hierarchical clustering differentiated HCC and CLD cases. Most HCC cases with advanced disease were clustered together and formed

Fragmentation reactions of protonated α-amino acids (AAs) were studied previously using tandem mass spectrometry (MS/MS) of unit mass resolution. Isobaric fragmentation products and minor fragmentation products could have been overlooked or misannotated. In the present study, we examined the fragmentation patterns of 19 AAs using high-resolution electrospray ionization MS/MS (HR-ESI-MS/MS) with collision-induced dissociation (CID). Isobaric fragmentation products from protonated Met and Trp were resolved and identified for the first time. Previously unreported fragmentation products from protonated Met, Cys, Gln, Arg, and Lys were observed. Additionally, the chemical identity of a fragmentation product from protonated Trp that was incorrectly annotated in previous investigations was corrected. All previously unreported fragmentation products and reactions were verified by pseudo MS 3 experiments and/or MS/MS analyses of deuterated AAs. Clearer pictures of the fragmentation reactions for Met, Cys, Trp, Gln, Arg and Lys were obtained in the present study.

Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry, or surface-enhanced laser desorption/ionization ProteinChip technology, has been widely used in obtaining the quantitative profiles of tissue proteomes, particularly plasma proteomes. Its high-throughput nature and simplicity in its experimental procedures have allowed this technology to become a popular research tool for biomarker discovery in the past 5 years. After accumulating more research experiences, researchers now have a better understanding of the characteristics and limitations of this technology, as well as the pitfalls in biomarker research, by undertaking a comparative proteomic approach. This review provides an overview of the surface-enhanced laser desorption/ionization time-of-flight mass spectrometry, discusses its limitations and provides some possible solutions to help apply this technology to biomarker research.