Capillary LC−MS<sup>2</sup>at the Attomole Level for Monitoring and Discovering Endogenous Peptides in Microdialysis Samples Collected in Vivo

Haskins, William E.; Wang, Ziqiang; Watson, Christopher J.; Rostand, Rebecca R.; Witowski, Steven R.; Powell, David H.; Kennedy, Robert T.

doi:10.1021/ac010774d

Cited by 133 publications

(156 citation statements)

References 28 publications

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“…The combination of this sampling method with mass spectrometry facilitates investigation of the brain secretome in vivo. However, because of the low concentration of proteins in dialysate, which makes investigations challenging in terms of sensitivity, few studies have combined in vivo brain microdialysis and proteomics/peptidomics analysis (13)(14)(15)(16).…”

mentioning

confidence: 99%

Discovering New Bioactive Neuropeptides in the Striatum Secretome Using in Vivo Microdialysis and Versatile Proteomics

Bernay

Gaillard

Guryča

et al. 2009

Molecular & Cellular Proteomics

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The striatum, a major component of the brain basal nuclei, is central for planning and executing voluntary movements and undergoes lesions in neurodegenerative disorders such as Huntington disease. To perform highly integrated tasks, the striatum relies on a complex network of communication within and between brain regions with a key role devoted to secreted molecules. To characterize the rat striatum secretome, we combined in vivo microdialysis together with proteomics analysis of trypsin digests and peptidomics studies of native fragments. This versatile approach, carried out using different microdialysis probes and mass spectrometer devices, allowed evidencing with high confidence the expression of 88 proteins and 100 processed peptides. Their secretory pathways were predicted by in silico analysis. Whereas high molecular weight proteins were mainly secreted by the classical mode (94%), low molecular weight proteins equally used classical and non-classical modes (53 and 47%, respectively). In addition, our results suggested alternative secretion mechanisms not predicted by bioinformatics tools. Based on spectrum counting, we performed a relative quantification of secreted proteins and peptides in both basal and neuronal depolarization conditions. This allowed detecting a series of neuropeptide precursors and a 6-fold increase for neurosecretory protein VGF and In mammalian brain, the striatum plays a critical role for planning and executing voluntary movements and is also involved in cognitive processes (1). The striatum makes use of a complex network architecture connecting specialized anatomical structures to achieve these highly integrated tasks. It receives projections from primary sensory and motor cortices as well as motor thalamic nuclei and sends projections to downstream basal ganglia structures, thereby influencing the control of cortical and brainstem motor systems (2). In this context, communication within and between brain structures appears as a key element for brain functioning. For cell-to-cell communication, secreted proteins play a pivotal regulatory role. To enter the secretory pathway, it has been long assumed that an N-terminal signal peptide sequence is strictly required. However, recent studies have shown that endoplasmic reticulum-and Golgi-independent or non-classical mechanisms may be responsible for protein secretion (3). The extracellular medium is thus more complex than previously suspected, and its characterization has gained a special interest (4, 5). In silico analyses suggest that mature proteins secreted via classical and non-classical mechanisms share common physicochemical properties (6). In this respect, proteomics is a powerful approach for systematically analyzing proteins present in the extracellular medium (7-9). For neurochemical monitoring of the secretome within the brain, only a few tools provide an appropriate insight into its spatial and temporal dynamics. Microdialysis, in particular, has been shown to be a powerful tool for exploring the extracellular content o...

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mentioning

confidence: 99%

Discovering New Bioactive Neuropeptides in the Striatum Secretome Using in Vivo Microdialysis and Versatile Proteomics

Bernay

Gaillard

Guryča

et al. 2009

Molecular & Cellular Proteomics

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“…To improve the sensitivity for monitoring in vivo peptide secretion, Kennedy and colleagues (75,76) employed capillary LC columns (25-μm inner diameter) interfaced to a quadrupole ion trap mass spectrometer. The miniaturization of the LC column, coupled with a smaller electrospray emitter and automated two-pump system for high flow-rate sample loading and low flow-rate peptide elution, enabled the detection of attomole-level peptides and the identification of close to 30 SPs in brain extracellular fluid collected in vivo from live rats.…”

Section: Mass Spectrometric Analysis Of Tissue Extracts and Extracellmentioning

confidence: 99%

“…Because the peptides are released from a cell/neurohemal release area and are rapidly diluted by diffusion from localized release sites, the concentrations are often in the picomolar-to-nanomolar range, with sample volumes in the nanoliter-to-microliter range. Thus, the collected material is often introduced to capillary-scale separations and requires high-sensitivity MS (75,76,140,143,144).…”

Section: Peptide Function: Dynamic Measurementsmentioning

confidence: 99%

Peptides in the Brain: Mass Spectrometry–Based Measurement Approaches and Challenges

Li¹,

Sweedler

2008

Annual Rev. Anal. Chem.

131

172

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The function and activity of almost every circuit in the human brain are modified by the signaling peptides (SPs) surrounding the neurons. As the complement of peptides can vary even in adjacent neurons and their physiological actions can occur over a broad range of concentrations, the required figures of merit for techniques to characterize SPs are surprisingly stringent. In this review, we describe the formation and catabolism of SPs and highlight a range of mass spectrometric techniques used to characterize SPs. Approaches that supply high chemical information content, direct tissue profiling, spatially resolved data, and temporal information on peptide release are also described. Because of advances in measurement technologies, our knowledge of SPs has greatly increased over the last decade, and SP discoveries will continue as the capabilities of modern measurement approaches improve. 451

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“…Trypsin was added at a 50:1 ratio to the sample, and the proteins were digested overnight at 37°C. Tryptic peptides were analyzed by capillary liquid chromatography-tandem mass spectrometry (CLC-MS 2 ) with a system similar to that described elsewhere (13). Sequence information was obtained for tryptic peptides via collision-induced dissociation.…”

Section: S Rdna Sequencingmentioning

confidence: 99%

Species Differentiation of a Diverse Suite of Bacillus Spores by Mass Spectrometry-Based Protein Profiling

Dickinson¹,

Duc

Haskins

et al. 2004

Appl Environ Microbiol

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In this study, we demonstrate the versatility of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOFMS) protein profiling for the species differentiation of a diverse suite of Bacillus spores. MALDI-TOFMS protein profiles of 11 different strains of Bacillus spores, encompassing nine different species, were evaluated. Bacillus species selected for MALDI-TOFMS analysis represented the sporeforming bacterial diversity of typical class 100K clean room spacecraft assembly facilities. A one-step sample treatment and MALDI-TOFMS preparation were used to minimize the sample preparation time. A library of MALDI-TOFMS spectra was created from these nine Bacillus species, the most diverse protein profiling study of the genus reported to date. Linear correlation analysis was used to successfully differentiate the MALDI-TOFMS protein profiles from all strains evaluated in this study. The MALDI-TOFMS protein profiles were compared with 16S rDNA sequences for their bacterial systematics and molecular phylogenetic affiliations. The MALDI-TOFMS profiles were found to be complementary to the 16S rDNA analysis. Proteomic studies of Bacillus subtilis 168 were pursued to identify proteins represented by the biomarker peaks in the MALDI-TOFMS spectrum. Four small, acid-soluble proteins (A, B, C, and D), one DNA binding protein, hypothetical protein ymf J, and four proteins associated with the spore coat and spore coat formation (coat JB, coat F, coat T, and spoIVA) were identified. The ability to visualize higher-molecular-mass coat proteins (10 to 25 kDa) as well as smaller proteins (<10 kDa) with MALDI-TOFMS profiling is critical for the complete and effective species differentiation of the Bacillus genus.Rapid, sensitive, and selective microbial detection and identification at the species and strain level are necessary to differentiate between viable pathogenic and nonpathogenic microbial species. The development of technology to accomplish this level of distinction for microbial species would have a significant impact on occupational and health care, homeland defense, and environmental monitoring. For over a century, microbial identification techniques have depended on conventional culture-based methods that characterize phenotypic differences and rely on biochemical and morphological tests. These methods are time-consuming and laborious, and the results are often subjective (38,41). In order to overcome the problems involved with phenotypic characterization, 16S rRNA analysis has been used for decades to more accurately define the phylogenetic affiliation of the given test microorganism (29). However, being highly conserved, the 16S rRNA molecule at times cannot differentiate closely related microbial species (41, 43). Therefore, alternative biomarkers (44) or a suite of protein profiling methods would be useful in order to effectively differentiate closely related microbial species.Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOFMS)-based microbial detec...

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Capillary LC−MS²at the Attomole Level for Monitoring and Discovering Endogenous Peptides in Microdialysis Samples Collected in Vivo

Cited by 133 publications

References 28 publications

Discovering New Bioactive Neuropeptides in the Striatum Secretome Using in Vivo Microdialysis and Versatile Proteomics

Discovering New Bioactive Neuropeptides in the Striatum Secretome Using in Vivo Microdialysis and Versatile Proteomics

Peptides in the Brain: Mass Spectrometry–Based Measurement Approaches and Challenges

Species Differentiation of a Diverse Suite of Bacillus Spores by Mass Spectrometry-Based Protein Profiling

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Capillary LC−MS2at the Attomole Level for Monitoring and Discovering Endogenous Peptides in Microdialysis Samples Collected in Vivo

Cited by 133 publications

References 28 publications

Discovering New Bioactive Neuropeptides in the Striatum Secretome Using in Vivo Microdialysis and Versatile Proteomics

Discovering New Bioactive Neuropeptides in the Striatum Secretome Using in Vivo Microdialysis and Versatile Proteomics

Peptides in the Brain: Mass Spectrometry–Based Measurement Approaches and Challenges

Species Differentiation of a Diverse Suite of Bacillus Spores by Mass Spectrometry-Based Protein Profiling

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Capillary LC−MS²at the Attomole Level for Monitoring and Discovering Endogenous Peptides in Microdialysis Samples Collected in Vivo