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We present a new development of the Tag-Mass concept based on a photocleavable linker with tagged molecules for polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) quantification coupled to mass spectrometry. PCR-MS and immunosorbent assay-MS with tagged oligonucleotides, bases, and antibodies will allow the acquisition of multiplexed information from genomic, transcriptomic, and proteomic experiments. This is a novel application of Tag-Mass from tissue imaging to fluid quantification and will open doors to several clinical applications ranging from biomarker-driven gene modulation to use at the patient's bedside following treatment. 10.1021/ac901416s

Matrix-assisted laser desorption/ionization (MALDI) is a new tool that can acquire the localization of various compounds, including peptides and proteins, directly from tissue sections. Despite the important developments recently performed in the field of MALDI imaging in tissue, the precise identification of compounds still needs improvement. We have developed N-terminal chemical derivafization strategies to improve tissue identification of proteins, including de novo sequencing performance. We have first focused on sulfonation agents, such as 4-SPITC and 3-SBASE. These two derivatizations were optimized to be performed directly on tissue sections. By adding a negative charge at the N-terminus of a tryptic digest peptide, we were able to generate a complete y fragment series directly from the tissue. Of these derivatizations, 3-SBASE has shown to be more efficient, as loss of the derivative group is one of the major fragmentation pathways for 4-SPITC. 3-SBASE was optimized so that the derivatization reaction could be automatically performed using an automatic microspotting device. It was then included in an automatic process that included automated trypsin digestion and matrix deposition. Derivatizations allowed the acquisition to be easily interpretable by [MS.sup.2] spectra, leading to very precise identification as well as easy manual reading of sequences for de novo sequencing. It was observed that only arginine-termihated peptides were observed after derivatization, likely due to the high gas-phase basicity of such peptides compared to those that are lysine-terminated. We also observed a stop in the y fragmentation series for peptides presenting a miscleavage. We have now begun to study a different derivatization using N-succinimidyloxycarbonylmethyl)tris(2,4,6-trimethoxyphenyl) phosphonium bromide (TMPP). This derivatization allows the orientating of a fragmentation toward a series of fragment ions, and thus it is independent of the presence of basic residues in the sequence. This derivatization can be performed at room temperature, which greatly facilitates the automation of the process. The TMPP derivatization therefore yields an advantageous new generation of derivatives suited for use in tissue. 10.1021/ac901043n

Direct tissue analysis using MALDI-MS allows the generation of profiles while maintaining the integrity of the tissue, displaying cellular localizations and avoiding tedious extraction and purification steps. However, lower spectral quality can result from direct tissue analysis due to variations in section thickness, the nature of the tissue, and the limited access to peptides/proteins due to high lipid content. To improve signal sensitivity, we have developed a tissue-washing procedure using organic solvents traditionally used for lipid extraction, i.e., CH[Cl.sub.3], hexane, toluene, acetone, and xylene. The increased detection for peptides/proteins (m/z 5000-30 000) is close to 40% with chloroform or xylene, and 25% with hexane, while also improving sample reproducibility for each solvent used in the present study. This strategy improved matrix cocrystallization with tissue peptides/ proteins and more importantly with cytoplasmic proteins without delocalization. The extracted lipids were characterized by nanoESI-QqTOF/MS/MS using the precursor ion mode, lithium adducts, or both and were identified as phospholipids including phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and lysophos-phatidylinositol, confirming membrane lipid extraction from the tissues.

Direct analysis of tissue by MALDI-MS allows the acquisition of its biomolecular profile while maintaining the integrity of the tissue, giving cellular localization, and avoiding tedious extraction and purification steps. However, direct tissue analysis generally leads to some extent to a lowered spectral quality due to variation in thickness, freezing tissue date, and nature of the tissue. We present here new technical developments for the direct tissue analysis of peptides with ionic liquid made of matrix mixtures ([alpha]-cyano-4-hydroxycinnamic acid (CHCA)/2-amino-4-methyl-5-nitropyridine and [alpha]-cyano-4-hydroxy-cinnamic acid/N,N-dimethylaniline (CHCA/DANI)). The properties of these direct tissue analysis matrixes, especially CHCA/aniline when compared to CHCA, 2,5-dihydroxybenzoic acid, and sinapinic acid, are as follows: (1) better spectral quality in terms of resolution, sensitivity, intensity, noise, number of compounds detected, and contaminant tolerance, (2) better crystallization on tissues, i.e., coverage capacity, homogeneity of crystallization, homogeneity of crystal sizes, and time of crystallization, (3) better analysis duration in tenn of vacuum stability, (4) better resistance to laser irradiation especially for high-frequency lasers, (5) better ionic yield in negative mode, and (6) enough fragmentation yield to use the PSD mode on sections to get structural information. Applied to MALDI imaging on a MALDI LIFT-TOF with a 50-Hz laser frequency, these ionic matrixes have allowed the realization of a new type of image in both polarities and reflector mode using the same tissue section. These results give a new outlook on peptide tissue profiling by MS, characterization of compounds from tissue slices, and MALDI-MS high-quality imaging.

In Mammals, microglial cells are considered as the resident immune cells in central nervous system (CNS). Many studies demonstrated that, after injury, these cells are activated and recruited at the lesion site. Leech microglia present a similar pattern of microglial activation and migration upon experimental lesion of CNS. This activation is associated with the release of a large amount of extracellular vesicles (EVs). We collected EVs released by microglia primary culture and compared two different protocols of isolation: one with differential ultracentrifugation (UC) and one using an additional Optiprep™ Density Gradient (ODG) ultracentrifugation. Nanoparticles tracking analysis (NTA) and transmission electron microscopy (TEM) were used to assess vesicles size and morphology. The protein content of isolated EVs was assessed by mass spectrometry approaches. Results showed the presence of EV-specific proteins in both procedures. The extensive proteomic analysis of each single ODG fractions confirmed the efficiency of this protocol in limiting the presence of co-isolated proteins aggregates and other membranous particles during vesicles isolation. The present study permitted for the first time the characterisation of microglial EV protein content in an annelid model. Interestingly, an important amount of proteins found in leech vesicles was previously described in EV-specific databases. Finally, purified EVs were assessed for neurotrophic activity and promote neurites outgrowth on primary cultured neurons. [ABSTRACT FROM AUTHOR]

Mass spectrometry-based methods for prostate cancer biomarker discovery are hampered by their low-throughput capabilities because of extensive sample preparation. We present the parafilm-assisted microdissection technique coupled with label-free quantification and bioinformatics analysis as a means to evaluate directly protein expression changes on benign and tumor regions. [ABSTRACT FROM AUTHOR]

Spinal cord injury (SCI) has been implicated in neural cell loss and consequently functional motor and sensory impairment. In this study, we propose an alginate -based neurobridge enriched with/without trophic growth factors (GFs) that can be utilized as a therapeutic approach for spinal cord repair. The bioavailability of key GFs, such as Epidermal Growth factor (EGF) and basic Fibroblast Growth Factor (bFGF) released from injected alginate biomaterial to the central lesion site significantly enhanced the sparing of spinal cord tissue and increased the number of surviving neurons (choline acetyltransferase positive motoneurons) and sensory fibres. In addition, we document enhanced outgrowth of corticospinal tract axons and presence of blood vessels at the central lesion. Tissue proteomics was performed at 3, 7 and 10 days after SCI in rats indicated the presence of anti-inflammatory factors in segments above the central lesion site, whereas in segments below, neurite outgrowth factors, inflammatory cytokines and chondroitin sulfate proteoglycan of the lectican protein family were overexpressed. Collectively, based on our data, we confirm that functional recovery was significantly improved in SCI groups receiving alginate scaffold with affinity-bound growth factors (ALG +GFs), compared to SCI animals without biomaterial treatment. [ABSTRACT FROM AUTHOR]

Unlike mammals, the CNS of the medicinal leech can regenerate damaged neurites, thus restoring neural functions after lesion. We previously demonstrated that the injured leech nerve cord is able to mount an immune response promoting the regenerative processes. Indeed neurons and microglia express sensing receptors like Hm-TLR1, a leech TLR ortholog, associated with chemokine release in response to a septic challenge or lesion. To gain insights into the TLR signaling pathways involved during these neuroimmune responses, members of the MyD88 family were investigated. In the present study, we report the characterization of Hm-MyD88 and Hm-SARM. The expression of their encoding gene was strongly regulated in leech CNS not only upon immune challenge but also during CNS repair, suggesting their involvement in both processes. This work also showed for the first time that differentiated neurons of the CNS could respond to LPS through a MyD88-dependent signalling pathway, while in mammals, studies describing the direct effect of LPS on neurons and the outcomes of such treatment are scarce and controversial. In the present study, we established that this PAMP induced the relocalization of Hm-MyD88 in isolated neurons. [ABSTRACT FROM AUTHOR]

Cnidarian–dinoflagellate photosynthetic symbioses are fundamental to biologically diverse and productive coral reef ecosystems. The hallmark of this symbiotic relationship is the ability of dinoflagellate symbionts to supply their cnidarian host with a wide range of nutrients. Many aspects of this association nevertheless remain poorly characterized, including the exact identity of the transferred metabolic compounds, the mechanisms that control their exchange across the host–symbiont interface, and the precise subcellular fate of the translocated materials in cnidarian tissues. This lack of knowledge is mainly attributed to difficulties in investigating such metabolic interactions both in situ, i.e. on intact symbiotic associations, and at high spatial resolution. To address these issues, we illustrate the application of two in situ and high spatial resolution molecular and ion imaging techniques–matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) and the nano-scale secondary-ion mass spectrometry (NanoSIMS) ion microprobe. These imaging techniques provide important new opportunities for the detailed investigation of many aspects of cnidarian–dinoflagellate associations, including the dynamics of cellular interactions. [ABSTRACT FROM AUTHOR]