Hormone complement of the Cancer productus sinus gland and pericardial organ: an anatomical and mass spectrometric investigation.

In crustaceans, circulating hormones influence many physiological processes. Two neuroendocrine organs, the sinus gland (SG) and the pericardial organ (PO), are the sources of many of these compounds. As a first step in determining the roles played by hemolymph-borne agents in the crab Cancer productus, we characterized the hormone complement of its SG and PO. We show via transmission electron microscopy that the nerve terminals making up each site possess dense-core and/or electron-lucent vesicles, suggesting diverse complements of bioactive molecules for both structures. By using immunohistochemistry, we show that small molecule transmitters, amines and peptides, are among the hormones present in these tissues, with many differentially distributed between the two sites (e.g., serotonin in the PO but not the SG). With several mass spectrometric (MS) methods, we identified many of the peptides responsible for the immunolabeling and surveyed the SG and PO for peptides for which no antibodies exist. By using MS, we characterized 39 known peptides [e.g., beta-pigment-dispersing hormone (beta-PDH), crustacean cardioactive peptide, and red pigment-concentrating hormone] and de novo sequenced 23 novel ones (e.g., a new beta-PDH isoform and the first B-type allatostatins identified from a non-insect species). Collectively, our results show that diverse and unique complements of hormones, including many previously unknown peptides, are present in the SG and PO of C. productus. Moreover, our study sets the stage for future biochemical and physiological studies of these molecules and ultimately the elucidation of the role(s) they play in hormonal control in C. productus.

Studies on the substrate specificity of Escherichia coli galactokinase.

In vitro glycorandomization (IVG) technology is dependent upon the ability to rapidly synthesize sugar phosphates. Compared with chemical synthesis, enzymatic (kinase) routes to sugar phosphates would be attractive for this application. This work focuses upon the development of a high-throughput colorimetric galactokinase (GalK) assay and its application toward probing the substrate specificity and kinetic parameters of Escherichia coli GalK. The demonstrated dinitrosalicylic assay should also be generally applicable to a variety of sugar-processing enzymes. [reaction: see text]

Vaccine adjuvant activity of 3M-052: an imidazoquinoline designed for local activity without systemic cytokine induction.

The human Toll-like receptors (TLRs) are a family of receptors, which sense the presence of various structural elements of pathogens and damaged or effete components in the host. As they do so, they activate two critical arms of host defense, the rapid innate immune response and an adaptive immune response. The innate immune response is typified by the generation of Th1 cytokines, chemokines and type 1 interferons. As such, agonists for the TLRs have potential as antiviral and anticancer therapeutics. They are also well suited to function as vaccine adjuvants. 3M imidazoquinoline (IRM) molecules were the first synthetic small molecules identified as TLR agonists and can affect their biological activities through TLR7, TLR8, or both. The breadth of therapeutic opportunities for this family of molecules can require formulations tailored to the specific application. One consideration is specific formulations to avoid a systemic distribution of these TLR agonists and resulting cytokine storm-like effects on the host. 3M-052 is an IRM bearing a C18 lipid moiety and designed for slow dissemination from the site of application. In the present study 3M-052 has been evaluated for its in vitro TLR activity and for its efficacy as a vaccine adjuvant using a recombinant hemagglutinin from H1N1 A/Puerto Rico/8/34. Given subcutaneously, 3M-052 drives a strong Th1 response to hemagglutinin and serum neutralization of viable H1N1 A/Puerto Rico/8/34 virus in the absence of circulating TNFα or the induction of Th1 cytokines.

Prion disease diagnosis by proteomic profiling.

Definitive prion disease diagnosis is currently limited to postmortem assay for the presence of the disease-associated proteinase K-resistant prion protein. Using cerebrospinal fluid (CSF) from prion-infected hamsters, matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS), and support vector machines (SVM), we have identified peptide profiles characteristic of disease state. Using 10-fold leave-one-out cross-validation, we report a predictive accuracy of 72% with a true positive rate of 73% and a false positive rate of 27% demonstrating the suitability of using proteomic profiling and CSF for the development of multiple marker diagnostics of prion disease.

Combining MALDI-FTMS and bioinformatics for rapid peptidomic comparisons.

Increasing research efforts in large-scale mass spectral analyses of peptides and proteins have led to many advances in technology and method development for collecting data and improving the quality of data. However, the resultant large data sets often pose significant challenges in extracting useful information in a high-throughput manner. Here, we describe one such method where we analyzed a large mass spectral data set collected using decapod crustacean nervous tissue extracts separated via high-performance liquid chromatography (HPLC) coupled to high-resolution matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS). Following their acquisition, the data collected from discrete LC fractions was compiled and analyzed using an in-house developed software package that deisotoped, compressed, calibrated, and matched peaks to a list of known crustacean neuropeptides. By processing these data via bioinformatics tools such as hierarchical clustering, more than 110 neuropeptides that belong to 14 peptide families were mapped in five crustacean species. Overall, we demonstrate the utility of MALDI-FTMS in combination with a bioinformatics software package for the elucidation and comparison of peptidomes of varying crustacean species. This study established an effective methodology and will provide the basis for future investigations into more comprehensive comparative peptidomics with larger collection of species and phyla in order to gain a deeper understanding of the evolution and diversification of peptide families.

Imaging mass spectrometry of neuropeptides in decapod crustacean neuronal tissues.

Imaging mass spectrometry (IMS) of neuropeptides in crustacean neuronal tissues was performed on a MALDI-TOF/TOF instrument. Sample preparation protocols were developed for the sensitive detection of these highly complex endogenous signaling molecules. The neuromodulatory complements of the pericardial organ (PO) and brain of the Jonah crab, Cancer borealis, were mapped. Distributions of peptide isoforms belonging to 10 neuropeptide families were investigated using the IMS technique. Often, neuropeptides of high sequence homology were similarly located. However, two RFamide-family peptides and a truncated orcokinin peptide were mapped to locations distinct from other members of their respective families. Over 30 previously sequenced neuropeptides were identified based on mass measurement. For increased confidence of identification, select peptides were fragmented by post-source decay (PSD) and collisional-induced dissociation (CID). Collectively, this organ-level IMS study elucidates the spatial relationships between multiple neuropeptide isoforms of the same family as well as the relative distributions of neuropeptide families.

In situ tissue analysis of neuropeptides by MALDI FTMS in-cell accumulation.

Herein we report the first application of Fourier transform mass spectrometry for the analysis of neuropeptides directly from neuronal tissues. Sample preparation protocols and instrumentation conditions are developed to allow in situ neuropeptide analysis of the neuroendocrine organs freshly isolated from a marine organism Cancer borealis. The utility of a previously developed in-cell accumulation (ICA) technique is extended for peptide analysis in complex tissue samples. With the ICA procedure, ion signals from multiple laser shots are accumulated in the analyzer cell prior to detection. This procedure allows the accumulation of ion signals without accumulating noise, thus improving the signal-to-noise ratio and enhancing the sensitivity for the detection of trace-level endogenous neuropeptides. De novo sequencing of peptides directly from tissue samples becomes more feasible through this improvement. Additionally, an integrated pulse sequence is constructed to cover a wide mass range from m/z 215 to 9000 by centering quadrupole collection of ions at different masses for successive laser shots. Finally, improved mass measurement accuracy (2 ppm) for tissue peptide analysis is achieved using ICA by incorporating calibrants on a separate spot from the sample of interest without premixing calibration standards with the analytes.