Dataset Information

Moving Forward Real-Time Proteomics Mass Spectrometry by Remote Infrared Matrix-Assisted Laser Desorption-Ionization

ABSTRACT: Novel development makes remote real-time analysis with possible translation to in-vivo a reality. Remote Infrared Matrix Assisted Laser Desorption Ionization (Remote IR MALDI) system with endogenous water as matrix becomes real and allows to envisage real-time proteomics to be performed in the in-vivo context. Remote IR MALDI is demonstrated to be used to analyze peptides and proteins. Very interestingly, the corresponding mass spectra show ESI like charge states distribution, opening many applications for structural elucidation to be performed in real-time by Top-Down analysis. The charge states show no dependence toward laser wavelength or length of the transfer tube allowing for remote analyses to be perform 5 m away from the mass spectrometry (MS) instrument without modification of spectra. This brings also interesting features to the understanding of IR MALDI ionization mechanism

INSTRUMENT(S): Synapt MS

ORGANISM(S): Homo Sapiens (human) Bos Taurus (bovine)

TISSUE(S): Liver

SUBMITTER: Fournier Isabelle

LAB HEAD: Michel Salzet

PROVIDER: PXD008092 | Pride | 2018-04-20

REPOSITORIES: Pride

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Publications

In vivo Real-Time Mass Spectrometry for Guided Surgery Application.

Fatou Benoit B Saudemont Philippe P Leblanc Eric E Vinatier Denis D Mesdag Violette V Wisztorski Maxence M Focsa Cristian C Salzet Michel M Ziskind Michael M Fournier Isabelle I

Scientific reports 20160518

Here we describe a new instrument (SpiderMass) designed for in vivo and real-time analysis. In this instrument ion production is performed remotely from the MS instrument and the generated ions are transported in real-time to the MS analyzer. Ion production is promoted by Resonant Infrared Laser Ablation (RIR-LA) based on the highly effective excitation of O-H bonds in water molecules naturally present in most biological samples. The retrieved molecular patterns are specific to the cell phenotyp ...[more]

PMID: 27189490

Publication: 1/2

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Project description:Orchids are commercially important flowers, comprising one of the largest families of plants. Model plants such as Arabidopsis do not contain all plant genes. In this study, several molecular biology tools were integrated to isolate the flower-specific gene promoters of Oncidium Gower Ramsey (GR). A cDNA library of reproductive tissues was constructed and a microarray was established and used to compare gene expression in flowers and leaves. Five genes were highly expressed in flower tissues and the subcellular locations of the corresponding proteins were identified using fluorescent protein-fusion protein lip transient transformation, and related to their putative functions. BAC clones of the five genes, together with nine previously published flower and reproductive growth specific genes in Onc. GR, were identified and promoter regions were cloned by screening an Onc. GR BAC library. Interestingly, three of the five novel flower-abundant genes were putative trypsin inhibitor (TI) genes, OnTI1, OnTI2 and OnTI3, which were tandemly duplicated in the same BAC clone. Their promoters were identified using transient GUS reporter gene transformation and stable Arabidopsis transformation analyses. In conclusion, a combination of tools including a cDNA microarray, a BAC library, and a bombardment assay can be successfully employed to identify orchid genes and promoters. A total of 960 cDNAs derived from the Onc. GR flower cDNA library were amplified using PCR incorporating the T3 and M13 reverse universal primers. The PCR products were purified using the QIAquick PCR Purification kit (Qiagen, Germantown, MD) and eluted into 100 M-BM-5l of 0.1M-CM-^W Tris-EDTA buffer, pH 8.0. Purified PCR products were printed on GAPS II-coated slides (Corning, New York, NY) using the OmniGrid 100 microarray (Genomic Solutions, Ann Arbor, MI), and arranged into 1.8 M-CM-^W 1.8-cm arrays (spot size: 100 M-NM-<m) twice. These DNAs were cross-linked to the slide by baking the array at 80M-BM-0C for 2 h. After printing, the slides were left to dry overnight. The total RNA from leaves and flowers (25 M-NM-<g) was used for cDNA synthesis with either Cy3 or Cy5 dye molecules, using the 3DNA Expression Array Detection kit for microarrays (Array 50, version 2, Genisphere, Hatfield, PA). cDNA hybridization and washing procedures were performed according to the manufacturer's instructions. All experiments were carried out in triplicate, with repeats of each dye combination (n = 3).

Project description:The Ca2+/calmodulin-dependent kinase II is expressed in smooth muscle and believed to mediate intracellular calcium handling and calcium-dependent gene transcription. CaMKII is activated by Angiotensin-II. The multifunctional calcium/calmodulin-dependent kinase II (CaMKII) is activated by Angiotensin-II (Ang-II) in vascular smooth muscle cells (VSMC), but its impact on hypertension remains unknown. In our transgenic mice that express the inhibitor peptide CaMKIIN in smooth muscle (TG SM-CaMKIIN), the blood pressure response to chronic Ang-II infusion was significantly reduced as compared to littermate controls. Surprisingly, examination of blood pressure and heart rate under ganglionic blockade revealed a key role for VSMC CaMKII in efferent sympathetic outflow in response to Ang II hypertension. Consistently, the efferent splanchnic nerve activity and plasma phenylephrine concentrations were significantly lower in TG SM-CaMKIIN mice as compared to littermates. Moreover, the aortic depressor nerve activity was reset in hypertensive wild type animals, but not in TG SM-CaMKIIN mice, suggesting that changes in baroreceptor wall activity may be responsible for the blood pressure difference in Ang-II hypertension. The pulse wave velocity, a measure of vascular wall stiffness in vivo, was increased in aortas of hypertensive compared to normotensive WT animals. However, Ang-II infusion did not alter the pulse wave velocity in transgenic mice, suggesting that CaMKII in VSMC controls structural smooth muscle genes. Accordingly, analysis of gene expression changes in aortas from wild type and TG SM-CaMKIIN hypertensive mice demonstrated that CaMKII inhibition mainly altered the expression of muscle contractile proteins. In contrast, TG SM-CaMKIIN aortas were protected from the Ang-II induced upregulation of genes linked to proliferation, suggesting that CaMKII inhibition prevents the Ang-II-induced reprogramming of smooth muscle cell gene expression towards a proliferative phenotype. 5 WT C57Bl/6 and 5 mice that express the Ca2+/calmodulin-dependent kinase II peptide inhibitor CaMKIIN in smooth muscle only (TG SM-CaMKIIN) were infused with 1.25 ug/kg/min Angiotensin-II by osmotic minipump for 14 days. 5 WT and 5 transgenic mice infused with normal saline served as controls. The mice were sacrificed on day 14 and the thoracic aortas isolated. RNA was isolated and pooled for the following groups: WT (wild type), C (TG SM-CaMKIIN), WT-A (WT with Angiotensin-II), C-A (TG SM-CaMKIIN + Angiotensin-II)

Dataset Information

Moving Forward Real-Time Proteomics Mass Spectrometry by Remote Infrared Matrix-Assisted Laser Desorption-Ionization

Publications

In vivo Real-Time Mass Spectrometry for Guided Surgery Application.

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