Image-guided MALDI mass spectrometry for high-throughput single-organelle characterization

AbstractPeptidergic dense-core vesicles are involved in packaging and releasing neuropeptides and peptide hormones—critical processes underlying brain, endocrine and exocrine function. Yet, the heterogeneity within these organelles, even for morphologically defined vesicle types, is not well characterized because of their small volumes. We present image-guided, high-throughput mass spectrometry-based protocols to chemically profile large populations of both dense-core vesicles and lucent vesicles for their lipid and peptide contents, allowing observation of the chemical heterogeneity within and between these two vesicle populations. The proteolytic processing products of four prohormones are observed within the dense-core vesicles, and the mass spectral features corresponding to the specific peptide products suggest three distinct dense-core vesicle populations. Notable differences in the lipid mass range are observed between the dense-core and lucent vesicles. These single-organelle mass spectrometry approaches are adaptable to characterize a range of subcellular structures.

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Mass Spectrometry Imaging Using Dynamically Harmonized FT-ICR at Million Resolving Power: Rationalizing and Optimizing Sample Preparation and Instrumental Parameters

10.26434/chemrxiv.13013900 ◽

2020 ◽

Author(s):

Mathieu Tiquet ◽

Raphaël La Rocca ◽

Daan van Kruining ◽

Pilar Martinez-Martinez ◽

Gauthier Eppe ◽

...

Keyword(s):

Mass Spectrometry ◽

High Resolution ◽

Mass Spectrometry Imaging ◽

Mass Range ◽

Maldi Mass Spectrometry ◽

Mass Accuracy ◽

Ion Current ◽

Resolving Power ◽

Lipid Mass ◽

The Stability

<p><i>MALDI mass spectrometry imaging (MSI) is a powerful analytical method giving access to the 2D localizations of compounds in a thin section of a sample. To properly discern isobaric compounds in complex biological samples, dynamically harmonized ICR cell (ParaCell©) has been introduce to achieve extreme spectral resolution. However, high resolution MS images realized on a 9.4T FTICR High resolution instrument with recommended parameters suffered from an abnormal shifting of m/z ratios pixel to pixel. Resulting datasets show poor mass accuracy measurements and resolutions under estimations. By following the behavior of the Total Ion Current in function of the number of laser shots, the abnormal mass shifting phenomenon has been linked to the stability of the Total Ion Current (TIC) during images acquisitions. An optimization of laser parameters is proposed in order to limit the observed mass shift to retain machine specifications during MSI analyses. It is also shown that the method has been successfully employed to realize quality MS images with resolution above 1,000,000 in the lipid mass range across the whole image.</i></p>

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High-Throughput Monitoring of Multiclass Syrup Adulterants in Honey Based on the Oligosaccharide and Polysaccharide Profiles by MALDI Mass Spectrometry

Journal of Agricultural and Food Chemistry ◽

10.1021/acs.jafc.9b05317 ◽

2019 ◽

Vol 67 (40) ◽

pp. 11256-11261 ◽

Cited By ~ 9

Author(s):

Liangliang Qu ◽

Yuming Jiang ◽

Xueyong Huang ◽

Meng Cui ◽

Fangjian Ning ◽

...

Keyword(s):

Mass Spectrometry ◽

High Throughput ◽

Maldi Mass Spectrometry

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MicroRNA exocytosis by large dense-core vesicle fusion

10.1101/093278 ◽

2016 ◽

Author(s):

Alican Gümürdü ◽

Ramazan Yildiz ◽

Erden Eren ◽

Gökhan Karakülah ◽

Turgay Ünver ◽

...

Keyword(s):

Next Generation Sequencing ◽

Vesicle Fusion ◽

Dense Core ◽

Snare Complex ◽

Dense Core Vesicle ◽

Next Generation ◽

Dense Core Vesicles ◽

Large Dense Core Vesicles ◽

Extracellular Mirna ◽

Generation Sequencing

AbstractNeurotransmitters and peptide hormones are secreted into outside the cell by a vesicle fusion process. Although non-coding RNA (ncRNA) that include microRNA (miRNA) regulates gene expression inside the cell where they are transcribed, extracellular miRNA has been recently discovered outside the cells, proposing that miRNA might be released to participate in cell-to-cell communication. Despite its importance of extracellular miRNA, the molecular mechanisms by which miRNA can be stored in vesicles and released by vesicle fusion remain enigmatic. Using next-generation sequencing, vesicle purification techniques, and synthetic neurotransmission, we observe that large dense-core vesicles (LDCVs) contain a variety of miRNAs including miR-375. Furthermore, miRNA exocytosis is mediated by the SNARE complex and accelerated by Ca2+. Our results suggest that miRNA can be a novel neuromodulator that can be stored in vesicles and released by vesicle fusion together with classical neurotransmitters.One Sentence SummaryUsing next-generation sequencing (NGS) for microRNA (miRNA) and synthetic neurotransmission, we observed that large dense-core vesicles (LDCVs) contain a variety of miRNA together with classical neurotransmitters, and that miRNA can be released by vesicle fusion mediated by SNARE.

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The Slp4-a Linker Domain Controls Exocytosis through Interaction with Munc18-1·Syntaxin-1a Complex

Molecular Biology of the Cell ◽

10.1091/mbc.e05-11-1047 ◽

2006 ◽

Vol 17 (5) ◽

pp. 2101-2112 ◽

Cited By ~ 58

Author(s):

Takashi Tsuboi ◽

Mitsunori Fukuda

Keyword(s):

Plasma Membrane ◽

Specific Interaction ◽

Inhibitory Effect ◽

Neuroendocrine Cells ◽

Dense Core ◽

Dense Core Vesicle ◽

Dense Core Vesicles ◽

Syntaxin 1A ◽

Vesicle Exocytosis ◽

Linker Domain

Synaptotagmin-like protein 4-a (Slp4-a)/granuphilin-a is specifically localized on dense-core vesicles in certain neuroendocrine cells and negatively controls dense-core vesicle exocytosis through specific interaction with Rab27A. However, the precise molecular mechanism of its inhibitory effect on exocytosis has never been elucidated and is still a matter of controversy. Here we show by deletion and chimeric analyses that the linker domain of Slp4-a interacts with the Munc18-1·syntaxin-1a complex by directly binding to Munc18-1 and that this interaction promotes docking of dense-core vesicles to the plasma membrane in PC12 cells. Despite increasing the number of plasma membrane docked vesicles, expression of Slp4-a strongly inhibited high-KCl–induced dense-core vesicle exocytosis. The inhibitory effect by Slp4-a is absolutely dependent on the linker domain of Slp4-a, because substitution of the linker domain of Slp4-a by that of Slp5 (the closest isoform of Slp4-a that cannot bind the Munc18-1·syntaxin-1a complex) completely abrogated the inhibitory effect. Our findings reveal a novel docking machinery for dense-core vesicle exocytosis: Slp4-a simultaneously interacts with Rab27A and Munc18-1 on the dense-core vesicle and with syntaxin-1a in the plasma membrane.

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High-Throughput Method for N-Terminal Sequencing of Proteins by MALDI Mass Spectrometry

Analytical Chemistry ◽

10.1021/ac048776w ◽

2005 ◽

Vol 77 (2) ◽

pp. 645-651 ◽

Cited By ~ 38

Author(s):

Minoru Yamaguchi ◽

Takashi Nakazawa ◽

Hiroki Kuyama ◽

Takashi Obama ◽

Eiji Ando ◽

...

Keyword(s):

Mass Spectrometry ◽

High Throughput ◽

Maldi Mass Spectrometry ◽

High Throughput Method

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Atg16L1, an essential factor for canonical autophagy, participates in hormone secretion from PC12 cells independently of autophagic activity

Molecular Biology of the Cell ◽

10.1091/mbc.e12-01-0010 ◽

2012 ◽

Vol 23 (16) ◽

pp. 3193-3202 ◽

Cited By ~ 44

Author(s):

Koutaro Ishibashi ◽

Takefumi Uemura ◽

Satoshi Waguri ◽

Mitsunori Fukuda

Keyword(s):

Pc12 Cells ◽

Secretory Pathway ◽

Biological Activities ◽

Hormone Secretion ◽

Cell Types ◽

Small Gtpase ◽

Dense Core ◽

Dense Core Vesicle ◽

Dense Core Vesicles ◽

Autophagic Activity

Autophagy is a bulk degradation system in all eukaryotic cells and regulates a variety of biological activities in higher eukaryotes. Recently involvement of autophagy in the regulation of the secretory pathway has also been reported, but the molecular mechanism linking autophagy with the secretory pathway remains largely unknown. Here we show that Atg16L1, an essential protein for canonical autophagy, is localized on hormone-containing dense-core vesicles in neuroendocrine PC12 cells and that knockdown of Atg16L1 causes a dramatic reduction in the level of hormone secretion independently of autophagic activity. We also find that Atg16L1 interacts with the small GTPase Rab33A and that this interaction is required for the dense-core vesicle localization of Atg16L1 in PC12 cells. Our findings indicate that Atg16L1 regulates not only autophagy in all cell types, but also secretion from dense-core vesicles, presumably by acting as a Rab33A effector, in particular cell types.

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Putative Protein Biomarkers of Escherichia coli Antibiotic Multiresistance Identified by MALDI Mass Spectrometry

Biology ◽

10.3390/biology9030056 ◽

2020 ◽

Vol 9 (3) ◽

pp. 56 ◽

Cited By ~ 1

Author(s):

Telma de Sousa ◽

Didier Viala ◽

Laetitia Théron ◽

Christophe Chambon ◽

Michel Hébraud ◽

...

Keyword(s):

Mass Spectrometry ◽

Escherichia Coli ◽

Antibiotic Resistance ◽

Mass Range ◽

Maldi Mass Spectrometry ◽

Extended Spectrum Beta Lactamase ◽

E Coli ◽

Machine Learning Classification ◽

Linear Mode ◽

Maldi Tof

The commensal bacteria Escherichia coli causes several intestinal and extra-intestinal diseases, since it has virulence factors that interfere in important cellular processes. These bacteria also have a great capacity to spread the resistance genes, sometimes to phylogenetically distant bacteria, which poses an additional threat to public health worldwide. Here, we aimed to use the analytical potential of MALDI-TOF mass spectrometry (MS) to characterize E. coli isolates and identify proteins associated closely with antibiotic resistance. Thirty strains of extended-spectrum beta-lactamase producing E. coli were sampled from various animals. The phenotypes of antibiotic resistance were determined according to Clinical and Laboratory Standards Institute (CLSI) methods, and they showed that all bacterial isolates were multi-resistant to trimethoprim-sulfamethoxazole, tetracycline, and ampicillin. To identify peptides characteristic of resistance to particular antibiotics, each strain was grown in the presence or absence of the different antibiotics, and then proteins were extracted from the cells. The protein fingerprints of the samples were determined by MALDI-TOF MS in linear mode over a mass range of 2 to 20 kDa. The spectra obtained were compared by using the ClinProTools bioinformatics software, using three machine learning classification algorithms. A putative species biomarker was also detected at a peak m/z of 4528.00.

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EIPR1 controls dense-core vesicle cargo retention and EARP complex localization in insulin-secreting cells

Molecular Biology of the Cell ◽

10.1091/mbc.e18-07-0469 ◽

2020 ◽

Vol 31 (1) ◽

pp. 59-79 ◽

Cited By ~ 1

Author(s):

Irini Topalidou ◽

Jérôme Cattin-Ortolá ◽

Blake Hummer ◽

Cedric S. Asensio ◽

Michael Ailion

Keyword(s):

Insulin Secretion ◽

Dense Core ◽

Dense Core Vesicle ◽

Secretory Vesicles ◽

Dense Core Vesicles ◽

Interacting Protein ◽

Insulin Secreting Cells

Dense-core vesicles (DCVs) are secretory vesicles that package and secrete cargoes like insulin, but how cargo is sorted to DCVs is poorly understood. Here, it is shown that the EARP complex-interacting protein EIPR1 controls insulin secretion and localization of DCV cargo in insulin-secreting cells.

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Expression of mutant ELH prohormones in AtT-20 cells: the relationship between prohormone processing and sorting.

The Journal of Cell Biology ◽

10.1083/jcb.121.1.11 ◽

1993 ◽

Vol 121 (1) ◽

pp. 11-21 ◽

Cited By ~ 21

Author(s):

L J Jung ◽

T Kreiner ◽

R H Scheller

Keyword(s):

Secretory Pathway ◽

Egg Laying ◽

Proteolytic Processing ◽

Dense Core ◽

Dense Core Vesicles ◽

Basic Residue ◽

Amino Terminal ◽

Prohormone Processing ◽

Specific Order ◽

The Relationship

Posttranslational processing of many proteins is essential to the synthesis of fully functional molecules. The ELH (egg-laying hormone) prohormone is cleaved by endoproteases in a specific order at a variety of basic residue processing sites to produce mature peptides. The prohormone is first cleaved at a unique tetrabasic site liberating two intermediates (amino and carboxy) which are sorted to different classes of dense core vesicles in the bag cell neurons of Aplysia. When expressed in AtT-20 cells, the ELH prohormone is also first cleaved at the tetrabasic site. The amino-terminal intermediate is then sorted to the constitutive pathway, and a portion of the carboxy-terminal intermediate is sorted to the regulated pathway. Here, we use mutant constructs of the ELH prohormone expressed in AtT-20 cells to examine the relationship between prohormone processing and consequent sorting. Prohormone which has a dibasic site in place of the tetrabasic site is processed and sorted similarly to wild type. Furthermore, mutant prohormone which lacks the tetrabasic site is processed at an alternative site comprising three basic residues. In these mutant prohormones, mature ELH is still produced and stored in dense core vesicles while amino-terminal products are constitutively secreted. However, deletion of the tetrabasic and tribasic sites results in the rerouting of the amino-terminal intermediate products from the constitutive pathway to the regulated secretory pathway. Thus, in the ELH prohormone, the location of the proteolytic processing events within the secretory pathway and the order of cleavages regulate the sorting of peptide products.

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