Small-Volume Analysis of Cell–Cell Signaling Molecules in the Brain

Romanova, Elena V.; Aerts, Jordan T; Croushore, Callie A.; Sweedler, Jonathan V.

doi:10.1038/npp.2013.145

Cited by 31 publications

(36 citation statements)

References 161 publications

(148 reference statements)

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“…Additionally, the throughput and resolving power of single cell approaches are still too low to meaningfully assess the response of collections of neurons in a given brain region to experimental conditions such as the chemical effect of a drug, especially when there is a requirement for multiple biologically independent subjects. There is no doubt that with the continued development of sequencing and mass-spectrometry based methods for low sample input, new and exciting biology will emerge that advances our understanding of transcriptional and translational programmes both within the cell and in cell-to-cell signalling 108,109 .…”

Section: Challenges In Assessing the Proteome(s) Of The Cnsmentioning

confidence: 99%

Decoding neuroproteomics: integrating the genome, translatome and functional anatomy

et al. 2014

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The immense inter- and intra-cellular heterogeneity of the central nervous system (CNS) presents major challenges for high-throughput *omic analyses. Transcriptional, translational, and post-translational regulatory events are localised to specific neuronal cell-types, or sub-cellular compartments, resulting in discrete patterns of protein expression and activity. A spatial and quantitative knowledge of the “neuroproteome” is therefore critical to understanding normal and pathological aspects of functional genomics and anatomy of the CNS. Improvements in mass-spectrometry allow profiling of proteins at sufficient depth to complement results from high-throughput genomic and transcriptomic assays. However, there are challenges in integrating proteomic data with other data modalities and even greater challenges obtaining comprehensive neuroproteomic data with cell-type specificity. Here we discuss how proteomics should be exploited to enhance high-throughput functional genomic analysis by tighter integration of data analyses. We also discuss experimental strategies to achieve finer cellular and sub-cellular resolution in transcriptomic and proteomic studies of neural tissues.

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Section: Challenges In Assessing the Proteome(s) Of The Cnsmentioning

confidence: 99%

Decoding neuroproteomics: integrating the genome, translatome and functional anatomy

et al. 2014

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“…A unique two-step strategy hyphenates MS to targeted chemical analysis of immunocytochemical-selected peptidergic neurons containing selected biomarkers of interest [76]. While robust, single-cell peptidomics is difficult to mainstream for comprehensive chemical analysis, primarily due to technical challenges in adapting chemically complex nanoliter-volume samples to compatible nanoseparation platforms and hyphenating those with MS [77]. Therefore, direct targeted MALDI MS continues to be the most successful approach for single-cell measurement.…”

Section: Choosing Your Peptidomics Modus Operandi: a Guide On Methodomentioning

confidence: 99%

Peptidomics for the discovery and characterization of neuropeptides and hormones

Romanova

Sweedler

2015

Trends in Pharmacological Sciences

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The discovery of neuropeptides as signaling molecules with paracrine or hormonal regulatory functions has led to trailblazing advances in physiology and fostered the characterization of numerous neuropeptide-binding G-protein coupled receptors (GPCRs) as potential drug targets. The impact on human health has been tremendous: approximately 30% of commercial drugs act via the GPCR pathway. However, about 25% of the GPCRs encoded by the mammalian genome still lack their pharmacological identity. Searching for the orphan GPCR endogenous ligands that likely are neuropeptides has proved to be a formidable task. Here we describe the mass spectrometry-based technologies and experimental strategies that have been successful in achieving high throughput characterization of endogenous peptides in nervous and endocrine systems. Keywordsbioinformatics; endogenous peptides; sequencing; mass spectrometry; nervous system; quantitation What is peptidomics?Genome and transcriptome sequencing is upon us, so why we are still looking for ways to identify bioactive peptides in living systems? Bold genomic research efforts have provided extraordinary insights into the inventory of GPCR-receptor genes, the most coveted targets in drug development [1]. Gene association and knockout studies have illuminated the roles of various peptide genes in pathological conditions and diseases in animal models [2,3]. Yet genetic investigations do not determine the actual peptide gene products, neuropeptides, and hormones that mediate vital body functions and complex behaviors. The immense challenge is due to the complexity of molecular readout; a single gene can produce many products as a result of single nucleotide polymorphisms, alternative gene splicing, post-translational processing of precursor proteins, and the addition of chemical post-translational Corresponding author: Sweedler, J.V., jsweedle@illinois.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. HHS Public Access Author ManuscriptAuthor Manuscript Author ManuscriptAuthor Manuscript modifications (PTMs) of cleaved peptides that oftentimes cannot be inferred from genomic data. Even annotating peptide coding genes and in silico neuropeptidome prediction requires specialized expertise and bioinformatics tools [4][5][6].At the dawn of the new millennium, the term 'peptidomics' (see the workflow shown in Figure 1A) was formally adopted to describe a method for high throughput, direct measurement and structural characterization of the endogenous peptides present in a given biological sample (see detailed historical review by Schrader and co...

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“…Here we highlight methods that are particularly well suited to such small volumes assays: direct single cell profiling using MALDI MS, followed by two high-throughput methods, microarray mass spectrometry (MAMS) and mass cytometry, and MALDI MS imaging (MSI). For more information on these and other technologies for single cell and other small volume investigations, the interested reader is referred to several recent reviews [6,12,33,34]. …”

Section: Ms-based Approachesmentioning

confidence: 99%

Mass spectrometry-based characterization of endogenous peptides and metabolites in small volume samples

Ong

Tillmaand

Makurath

et al. 2015

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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Technologies to assay single cells and their extracellular microenvironments are valuable in elucidating biological function, but there are challenges. Sample volumes are low, the physicochemical parameters of the analytes vary widely, and the cellular environment is chemically complex. In addition, the inherent difficulty of isolating individual cells and handling small volume samples complicates many experimental protocols. Here we highlight a number of mass spectrometry (MS)-based measurement approaches for characterizing the chemical content of small volume analytes, with a focus on methods used to detect intracellular and extracellular metabolites and peptides from samples as small as individual cells. MS has become one of the most effective means for analyzing small biological samples due to its high sensitivity, low analyte consumption, compatibility with a wide array of sampling approaches, and ability to detect a large number of analytes with different properties without preselection. Having access to a flexible portfolio of MS-based methods allows quantitative, qualitative, untargeted, targeted, multiplexed, spatially resolved investigations of single cells and their similarly scaled extracellular environments. Combining MS with on-line and off-line sample conditioning tools, such as microfluidic and capillary electrophoresis systems, significantly increases the analytical coverage of the sample’s metabolome and peptidome, and improves individual analyte characterization / identification. Small volume assays help to reveal the causes and manifestations of biological and pathological variability, as well as the functional heterogeneity of individual cells within their microenvironments and within cellular populations.

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Small-Volume Analysis of Cell–Cell Signaling Molecules in the Brain

Cited by 31 publications

References 161 publications

Decoding neuroproteomics: integrating the genome, translatome and functional anatomy

Decoding neuroproteomics: integrating the genome, translatome and functional anatomy

Peptidomics for the discovery and characterization of neuropeptides and hormones

Mass spectrometry-based characterization of endogenous peptides and metabolites in small volume samples

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