We are grateful to Jie Li (University of Maryland, College Park) for valuable discussions on embryonic dissociation and FACS. We thank Vi M. Quach and Camille Lombard-Banek for assistance with sample preparation and data collection in previous studies exemplifying the proteomic applications that are highlighted in this protocol. Parts of this work were supported by the National Science Foundation under award number IOS-1832968 CAREER (to P.N.), the National Institutes of Health under award number R35GM124755 (to P.N.), the University of Maryland-National Cancer Institute Partnership Program (to P.N.), and COSMOS Club Foundation research awards (to A.B.B. and L.R.P.).

The authors declare no competing interests.

This protocol enables the characterization of protein expression in identified cell lineages in embryos of the Xenopus species. Stemming from HRMS, the methodology combines exquisite specificity in molecular identification, capability for multi-protein detection without molecular probes (usually hundreds to thousands of different proteins), and a capability for quantification. Adaptability to classical tools and workflows in cell and developmental (neuro)biology expand HRMS proteomics to exciting applications, i...

Our understanding of cell differentiation and the genesis of tissues and organs is the result of decades of elaborate targeted screens of genes and their products. Increasing our knowledge of all the biomolecules and their quantities during important cellular events would help unravel molecular mechanisms that control the spatial and temporal patterning of the vertebrate body plan. Technologies enabling molecular amplification and sequencing are now able to routinely report on large numbers of genes and transcripts, supporting hypothesis-driven studies in basic biological and translational research. To understand developing systems, a complex relationship between transcription and translation advocates for direct analysis of multiple proteins and their post-translational modifications. Global proteomics using in vitro biological systems, such as induced pluripotent stem cells, began to delineate mechanisms of tissue induction1,2. In complex organisms, such as the vertebrate embryo, development relies on morphogen gradients in the context of space and time3. It follows that gaining knowledge of proteomic changes as cells differentiate to form specialized tissues, such as neural tissues, offers a key to unlock molecular programs controlling normal and defective development and guide next-generation therapeutics.
The vertebrate South African clawed frog (Xenopus laevis) is a well-established model in cell and developmental, neuro-, and regenerative biology. Sir John Gurdon&#39;s 2012 Nobel Prize in Physiology or Medicine4,5 for the discovery of pluripotency of the somatic nucleus highlighted the importance of this model for discoveries in basic and translational studies. Xenopus embryos develop externally to the mother, thus facilitating direct manipulation of cells, cell clones, and gene expression over various stages of development. Asymmetrical pigmentation and stereotypical cell divisions enabled the charting of reproducible fate maps from the 16-6 and 32-cell7,8 stage embryo. For high-resolution mass spectrometry (HRMS) based proteomics, additional advantages of the model include relatively large size (~1 mm in diameter), which yields abundant protein content for analysis (~130 &#181;g in early cleavage-stage embryos, ~10 &#181;g of protein content in single cells of the 16-cell embryo)9,10.
At present, HRMS is the leading technology of choice for detecting proteins. This technology enables direct, sensitive, and specific detection and quantification of multiple, usually hundreds-to-thousands of different proteins11. Bottom-up proteomics by HRMS involves a series of interconnected steps. Following extraction from the cell/tissue sample, proteins are digested with a proteolytic enzyme, such as trypsin (bottom-up proteomics). The resulting peptides are separated based on their different physicochemical properties, including hydrophobicity (reversed-phase liquid chromatography, LC), net charge (ion-exchange chromatography), size (size exclusion chromatography), or electrophoretic mobility (capillary electrophoresis, CE). Peptides are then charged (ionized), typically using electrospray ionization (ESI), and peptide ions are detected and sequenced via gas-phase fragmentation by tandem HRMS. The resulting peptide data are mapped to the proteome of the organism being studied. With protein-specific (proteotypic) peptide ion signal intensity correlating with concentration, protein quantification can be performed label-free or label-based (multiplexing quantitation). HRMS proteomics yields a rich resource of information on the molecular state of the system under study, allowing for the generation of hypotheses and follow-up functional studies.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63586/63586fig01v2.jpg" /> 
Figure 1: Spatiotemporally scalable proteomics enabling cell-lineage guided HRMS proteomics in the developing (frog) embryo. (A) Visualization of the specimen (1) using a stereomicroscope (2) for injection of an identified cell (inset), using a fabricated micropipette (3) under control by a translation-stage (4). (B) Subcellular sampling of the identified left D11 cell in a 16-cell embryo. (C)&#160;Dissection of a whole D11 cell from a 16-cell embryo. (D) Fluorescent (green) tracing of the left and right D111 progenies from a 32-cell embryo to guide dissection of the neural ectoderm (NE) in the gastrula (stage 10) and isolation of the descendent tissue from the tadpole using FACS. Scale bars: 200 &#181;m for embryos, 1.25 mm for the vial. Figures were adapted with permission from references15,19,21,59. <a href="https://www.jove.com/files/ftp_upload/63586/63586fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The protocol presented here enables HRMS-based quantification of large numbers of proteins in identified cells/tissues in developing X. laevis embryos. The approach builds on accurate cell identification, reproducible cell fate maps, and established methodologies to track cell lineages in this biological model6,7,8. As shown in Figure 1, we study proteomes from single cells by employing whole-cell dissection or capillary microsampling to aspirate cellular content. Monitoring the lineage of a cell permits us to study the spatiotemporal evolution of the proteome as cells form tissues during gastrulation. The cell progeny is fluorescently marked by injecting a fluorophore conjugated to inert dextran or mRNA for fluorescent protein (e.g., green fluorescent protein, or GFP). The labeled progeny is isolated at desired developmental time points. During gastrulation, cell clones that are tightly clustered may be isolated by dissection. After gastrulation, cell clones may be distributed within the embryo owing to migratory movements and can be isolated from dissociated tissues by fluorescence-activated cell sorting (FACS). Proteins in these cells and tissues are measured via bottom-up proteomics employing HPLC or CE for separation and ESI tandem HRMS for identification. Cell-lineage-guided HRMS proteomics is scalable to different cell sizes and lineages within the embryo and is specific, sensitive, and quantitative. Through select examples shown here, we also demonstrate this protocol to be scalable and broadly adaptable to different types of cells and cell lineages.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63586/63586fig02.jpg" /> 
Figure 2: The bioanalytical workflow. Micro-dissection and capillary aspiration, or FACS facilitated sampling of cellular and clonal protein content. Depletion of abundant yolk proteins and separation by capillary electrophoresis (CE) or nano-flow liquid chromatography (LC) enhanced identification (ID) sensitivity using electrospray ionization (ESI) high-resolution mass spectrometry (HRMS). Quantification revealed dysregulation, supplying new information for hypothesis-driven studies in conjunction with information available from gene ontology (GO). Figures were adapted with permission from reference15. <a href="https://www.jove.com/files/ftp_upload/63586/63586fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetonitrile (LC-MS-grade)</td>
			<td>Fisher Scientific</td>
			<td>A955</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>ThermoFisher Scientific</td>
			<td>R0492</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium bicarbonate</td>
			<td>Fisher Scientific</td>
			<td>A643-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Analytical Column</td>
			<td>Thermo Scientific</td>
			<td>164941</td>
			<td></td>
		</tr>
		<tr>
			<td>Analytical microbalance</td>
			<td>Mettler-Toledo</td>
			<td>XSE105DU</td>
			<td></td>
		</tr>
		<tr>
			<td>Automatic peptide fractionation platform</td>
			<td>Agilent</td>
			<td>1260 Infinity II</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate Capillaries</td>
			<td>Sutter Instruments Co.</td>
			<td>B100-50-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate Capillaries (for making Emmitters)</td>
			<td>Sutter Instruments</td>
			<td>B100-75-10</td>
			<td></td>
		</tr>
		<tr>
			<td>C18 spin columns (for desalting)</td>
			<td>ThermoFisher Scientific</td>
			<td>89870</td>
			<td></td>
		</tr>
		<tr>
			<td>Camera ro monitor electrospray</td>
			<td>Edmund Optics Inc.</td>
			<td>EO-2018C</td>
			<td></td>
		</tr>
		<tr>
			<td>Combretastatin A4</td>
			<td>Millipore Sigma</td>
			<td>C7744</td>
			<td></td>
		</tr>
		<tr>
			<td>Commercial CESI system</td>
			<td>AB SCIEX</td>
			<td>CESI</td>
			<td></td>
		</tr>
		<tr>
			<td>(Cyclohexylamino)-1-propanesulfonic acid (CAPS)</td>
			<td>VWR</td>
			<td>97061-492</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytochalasin D</td>
			<td>Millipore Sigma</td>
			<td>C8273</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran, Alexa Fluor 488; 10,000 MW, Anionic, Fixable</td>
			<td>ThermoFisher Scientific</td>
			<td>D22910</td>
			<td></td>
		</tr>
		<tr>
			<td>Diothiothreitol</td>
			<td>Fisher Scientific</td>
			<td>FERR0861</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-30</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Fisher Scientific</td>
			<td>AAJ62786AP</td>
			<td></td>
		</tr>
		<tr>
			<td>Epifluorescence light source</td>
			<td>Lumencore</td>
			<td>AURA III</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf LoBing microcentrifuge&#160; tubes: protein</td>
			<td>Fisher Scientific</td>
			<td>13-698-793</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic acid (LC-MS-grade)</td>
			<td>Fisher Scientific</td>
			<td>A117-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Freezer (-20 &#176;C)</td>
			<td>Fisher Scientific</td>
			<td>97-926-1&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Freezer (-80 &#176;C)</td>
			<td>Thermo Scientific</td>
			<td>TSX40086A</td>
			<td></td>
		</tr>
		<tr>
			<td>Fused silica capillary</td>
			<td>Molex</td>
			<td>1088150596</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat Block</td>
			<td>Benchmark</td>
			<td>BSH300</td>
			<td></td>
		</tr>
		<tr>
			<td>High pressure liquid Chromatography System</td>
			<td>ThermoFisher Scientific</td>
			<td>Dionex Ultimate 3000 RSLC nanosystem</td>
			<td></td>
		</tr>
		<tr>
			<td>High voltage power supply</td>
			<td>Spellman</td>
			<td>CZE1000R</td>
			<td></td>
		</tr>
		<tr>
			<td>High-resolution Mass Spectrometer</td>
			<td>ThermoFisher Scientific</td>
			<td>Orbitrap Fusion Lumos Tribrid Mass Spectrometer</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC caps</td>
			<td>Thermo Scientific</td>
			<td>C4013-40A</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC Vials</td>
			<td>Thermo Scientific</td>
			<td>C4013-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Illuminator e.g. Goosenecks</td>
			<td>Nikon</td>
			<td>C-FLED2</td>
			<td></td>
		</tr>
		<tr>
			<td>Ingenuity Pathway Analysis</td>
			<td>Qiagen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Iodoacetamide</td>
			<td>Fisher Scientific</td>
			<td>AC122275000</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol (LC-MS-grade)</td>
			<td>Fisher Scientific</td>
			<td>A456</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol (LC-MS-grade)</td>
			<td>Fisher Scientific</td>
			<td>A456-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcapillary puller</td>
			<td>Suttor Instruments</td>
			<td>P-2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Microinjector</td>
			<td>Warner Instrument, Handem, CT</td>
			<td>PLI-100A</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropippette puller</td>
			<td>Sutter Instruments Co.</td>
			<td>P-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>MS data analysis software, commercial</td>
			<td>ProteomeDiscoverer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MS data analysis software, opensource</td>
			<td>MaxQuant</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>non-idet 40 substitute</td>
			<td>Millipore Sigma</td>
			<td>11754599001</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish 60 mm and 80 mm</td>
			<td>Fisher Scientific</td>
			<td>S08184</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce 10 &#181;L bed Zip-tips (for desalting)</td>
			<td>ThermoFisher Scientific</td>
			<td>87782</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce bicinchoninic acid protein assay kit</td>
			<td>ThermoFisher Scientific</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce quantitative colorimetric peptide assay</td>
			<td>ThermoFisher Scientific</td>
			<td>23275</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce Trypsin Protease (MS Grade)</td>
			<td>Fisher Scientific</td>
			<td>PI90058</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein LoBind vials</td>
			<td>Eppendorf</td>
			<td>0030108434 
			, 0030108442</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated Centrifuge</td>
			<td>Eppendorf</td>
			<td>5430R</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated Incubator</td>
			<td>Thermo Scientific</td>
			<td>PR505755R/3721</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium isethionate</td>
			<td>Millipore Sigma</td>
			<td>220078</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium pyrophosphate</td>
			<td>Sigma Aldrich</td>
			<td>221368-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless steel BGE vial</td>
			<td>Custom-Built</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless steel sample vials</td>
			<td>Custom-Built</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope (objective 10x)</td>
			<td>Nikon</td>
			<td>SMZ 1270, SZX18</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>VWR</td>
			<td>97063-790</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe pumps (2)</td>
			<td>Harvard Apparatus</td>
			<td>704506</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes (gas-tight): 500&#8211;1000 &#181;L</td>
			<td>Hamilton</td>
			<td>1750TTL</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer pipettes (Plastic, disposable)</td>
			<td>Fisher Scientific</td>
			<td>13-711-7M</td>
			<td></td>
		</tr>
		<tr>
			<td>Trap Column</td>
			<td>Thermo Scientific</td>
			<td>164750</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl (1 M solution)</td>
			<td>Fisher Scientific</td>
			<td>AAJ22638AP</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum concentrator capable of operation at 4&#8211;10 &#176;C</td>
			<td>Labconco</td>
			<td>7310022</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex-mixer</td>
			<td>Benchmark</td>
			<td>BS-VM-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Water (LC-MS-grade)</td>
			<td>Fisher Scientific</td>
			<td>W6</td>
			<td></td>
		</tr>
		<tr>
			<td>Water (LC-MS-grade)</td>
			<td>Fisher Scientific</td>
			<td>W6</td>
			<td></td>
		</tr>
		<tr>
			<td>XYZ translation stage</td>
			<td>Thorlabs</td>
			<td>PT3</td>
			<td></td>
		</tr>
		<tr>
			<td>XYZ translation stage</td>
			<td>Custom-Built</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

cell-lineage guided mass spectrometry proteomics in the developing (frog) embryo

Characterization of molecular events as cells give rise to tissues and organs raises a potential to better understand normal development and design efficient remedies for diseases. Technologies enabling accurate identification and quantification of diverse types and large numbers of proteins would provide still missing information on molecular mechanisms orchestrating tissue and organism development in space and time. Here, we present a mass spectrometry-based protocol that enables the measurement of thousands of proteins in identified cell lineages in Xenopus laevis (frog) embryos. The approach builds on reproducible cell-fate maps and established methods to identify, fluorescently label, track, and sample cells and their progeny (clones) from this model of vertebrate development. After collecting cellular contents using microsampling or isolating cells by dissection or fluorescence-activated cell sorting, proteins are extracted and processed for bottom-up proteomic analysis. Liquid chromatography and capillary electrophoresis are used to provide scalable separation for protein detection and quantification with high-resolution mass spectrometry (HRMS). Representative examples are provided for the proteomic characterization of neural-tissue fated cells. Cell-lineage-guided HRMS proteomics is adaptable to different tissues and organisms. It is sufficiently sensitive, specific, and quantitative to peer into the spatio-temporal dynamics of the proteome during vertebrate development.

This protocol enabled the study of proteins in single cells and their lineages as they establish tissues in X. laevis embryos. Figure 1 illustrates one such application of the approach to study proteins in neural-tissue-fated cells and the newly induced neural ectoderm in the embryo. As shown in Figure 1A, the bioanalytical workflow integrated traditional tools of cell and developmental biology to identify, inject/aspirate cells, and collect specimens. ...

Watch this Scientific Journal Video about Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing Frog Embryo at JoVE.com

Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing Frog Embryo

Here we describe a mass spectrometry-based proteomic characterization of cell lineages with known tissue fates in the vertebrate Xenopus laevis embryo.

Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing (Frog) Embryo

Characterization of molecular events as cells give rise to tissues and organs raises a potential to better understand normal development and design ...

cell-lineage-guided-mass-spectrometry-proteomics-developing-frog

Research

JoVE Journal

Chemistry

1.6K Views.  University of Maryland. Here we describe a mass spectrometry-based proteomic characterization of cell lineages with known tissue fates in the vertebrate Xenopus laevis embryo.

Video: Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing Frog Embryo

Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS)

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. The approach offers an unbiased and in-depth analysis that can enable the development of diagnostic tests, novel therapies, and further our understanding of disease processes. The inherent chemical diversity of the metabolome creates significant analytical challenges and there is no single experimental approach that can detect all metabolites. Additionally, the biological variation in individual metabolism and the dependence of metabolism on environmental factors necessitates large sample numbers to achieve the appropriate statistical power required for meaningful biological interpretation. To address these challenges, this tutorial outlines an analytical workflow for large scale non-targeted metabolite profiling of serum by UPLC-MS. The procedure includes guidelines for sample organization and preparation, data acquisition, quality control, and metabolite identification and will enable reliable acquisition of data for large experiments and provide a starting point for laboratories new to non-targeted metabolite profiling by UPLC-MS.

Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry UPLC-MS

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. This article outlines a typical workflow utilized for non-targeted metabolite profiling of serum including sample organization and preparation, data acquisition, data analysis, quality control, and metabolite identification.

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to ...

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first separated into polar and non-polar fractions by a liquid-liquid phase extraction, and partially purified to facilitate downstream analysis. Both aqueous (polar metabolites) and organic (non-polar metabolites) phases of the initial extraction are processed to survey a broad range of metabolites. Metabolites are separated by different liquid chromatography methods based upon their partition properties. In this method, we present microflow ultra-performance (UP)LC methods, but the protocol is scalable to higher flows and lower pressures. Introduction into the mass spectrometer can be through either general or compound optimized source conditions. Detection of a broad range of ions is carried out in full scan mode in both positive and negative mode over a broad m/z range using high resolution on a recently calibrated instrument. Label-free differential analysis is carried out on bioinformatics platforms. Applications of this approach include metabolic pathway screening, biomarker discovery, and drug development.

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry UPLC-HRMS

Untargeted metabolomics provides a hypothesis generating snapshot of a metabolic profile. This protocol will demonstrate the extraction and analysis of metabolites from cells, serum, or tissue. A range of metabolites are surveyed using liquid-liquid phase extraction, microflow ultraperformance liquid chromatography/high-resolution mass spectrometry (UPLC-HRMS) coupled to differential analysis software.

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first ...

A Guided Materials Screening Approach for Developing Quantitative Sol-gel Derived Protein Microarrays

Microarrays have found use in the development of high-throughput assays for new materials and discovery of small-molecule drug leads. Herein we describe a guided material screening approach to identify sol-gel based materials that are suitable for producing three-dimensional protein microarrays. The approach first identifies materials that can be printed as microarrays, narrows down the number of materials by identifying those that are compatible with a given enzyme assay, and then hones in on optimal materials based on retention of maximum enzyme activity. This approach is applied to develop microarrays suitable for two different enzyme assays, one using acetylcholinesterase and the other using a set of four key kinases involved in cancer. In each case, it was possible to produce microarrays that could be used for quantitative small-molecule screening assays and production of dose-dependent inhibitor response curves. Importantly, the ability to screen many materials produced information on the types of materials that best suited both microarray production and retention of enzyme activity. The materials data provide insight into basic material requirements necessary for tailoring optimal, high-density sol-gel derived microarrays.

A guided material screening approach to develop sol-gel derived protein doped microarrays using an emerging pin-printing method of fabrication is described. This methodology is demonstrated through the development of acetylcholinesterase and multikinase microarrays, which are used for cost-effective small-molecule screening.

Microarrays have found use in the development of high-throughput assays for new materials and discovery of small-molecule drug leads. Herein we describe a ...

Analyzing Protein Dynamics Using Hydrogen Exchange Mass Spectrometry

All cellular processes depend on the functionality of proteins. Although the functionality of a given protein is the direct consequence of its unique amino acid sequence, it is only realized by the folding of the polypeptide chain into a single defined three-dimensional arrangement or more commonly into an ensemble of interconverting conformations. Investigating the connection between protein conformation and its function is therefore essential for a complete understanding of how proteins are able to fulfill their great variety of tasks. One possibility to study conformational changes a protein undergoes while progressing through its functional cycle is hydrogen-1H/2H-exchange in combination with high-resolution mass spectrometry (HX-MS). HX-MS is a versatile and robust method that adds a new dimension to structural information obtained by e.g. crystallography. It is used to study protein folding and unfolding, binding of small molecule ligands, protein-protein interactions, conformational changes linked to enzyme catalysis, and allostery. In addition, HX-MS is often used when the amount of protein is very limited or crystallization of the protein is not feasible. Here we provide a general protocol for studying protein dynamics with HX-MS and describe as an example how to reveal the interaction interface of two proteins in a complex. &#160;&#160;

Protein conformation and dynamics are key to understanding the relationship between protein structure and function. Hydrogen exchange coupled with high-resolution mass spectrometry is a versatile method to study the conformational dynamics of proteins as well as characterizing protein-ligand and protein-protein interactions, including contact interfaces and allosteric effects.

All cellular processes depend on the functionality of proteins. Although the functionality of a given protein is the direct consequence of its unique ...

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical (Methylation) and Physical (Mass Spectrometry, Nuclear Magnetic Resonance) Techniques

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans. De-starched wheat endosperm cell walls were isolated as an alcohol-insoluble residue (AIR)1 and sequentially extracted with water (W-sol Fr) and 1 M KOH containing 1% NaBH4 (KOH-sol Fr) as described by Ratnayake et al. (2014)2. Two different approaches (see summary in Figure 1) are adopted. In the first, intact W-sol AXs are treated with 2AB to tag the original RE backbone chain sugar residue and then treated with an endoxylanase to generate a mixture of 2AB-labelled RE and internal region reducing oligosaccharides, respectively. In a second approach, the KOH-sol Fr is hydrolyzed with endoxylanase to first generate a mixture of oligosaccharides which are subsequently labelled with 2AB. The enzymically released ((un)tagged) oligosaccharides from both W- and KOH-sol Frs are then methylated and the detailed structural analysis of both the native and methylated oligosaccharides is performed using a combination of MALDI-TOF-MS, RP-HPLC-ESI-QTOF-MS and ESI-MSn. Endoxylanase digested KOH-sol AXs are also characterized by nuclear magnetic resonance (NMR) that also provides information on the anomeric configuration. These techniques can be applied to other classes of polysaccharides using the appropriate endo-hydrolases.

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical Methylation and Physical Mass Spectrometry, Nuclear Magnetic Resonance Techniques

This protocol describes the specific techniques used for the structural characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans by tagging the RE with 2 aminobenzamide prior to enzymatic (endoxylanase) hydrolysis and then analysis of the resultant oligosaccharides using mass spectrometry (MS) and nuclear magnetic resonance (NMR).

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of ...

Visualization of Ambient Mass Spectrometry with the Use of Schlieren Photography

This manuscript outlines how to visualize mass spectrometry ambient ionization sources using schlieren photography. In order to properly optimize the mass spectrometer, it is necessary to characterize and understand the physical principles of the source. Most commercial ambient ionization sources utilize jets of nitrogen, helium, or atmospheric air to facilitate the ionization of the analyte. As a consequence, schlieren photography can be used to visualize the gas streams by exploiting the differences in refractive index between the streams and ambient air for visualization in real time. The basic setup requires a camera, mirror, flashlight, and razor blade. When properly configured, a real time image of the source is observed by watching its reflection. This allows for insight into the mechanism of action in the source, and pathways to its optimization can be elucidated. Light is shed on an otherwise invisible situation.

This paper presents a protocol for the visualization of gaseous streams of an ambient ionization source using schlieren photography and mass spectrometry.

This manuscript outlines how to visualize mass spectrometry ambient ionization sources using schlieren photography. In order to properly optimize the mass ...

Synthesis and Mass Spectrometry Analysis of Oligo-peptoids

Peptoids are sequence-controlled peptide-mimicking oligomers consisting of N-alkylated glycine units. Among many potential applications, peptoids have been thought of as a type of molecular information storage. Mass spectrometry analysis has been considered the method of choice for sequencing peptoids. Peptoids can be synthesized via solid phase chemistry using a repeating two-step reaction cycle. Here we present a method to manually synthesize oligo-peptoids and to analyze the sequence of the peptoids using tandem mass spectrometry (MS/MS) techniques. The sample peptoid is a nonamer consisting of alternating N-(2-methyloxyethyl)glycine (Nme) and N-(2-phenylethyl)glycine (Npe), as well as an N-(2-aminoethyl)glycine (Nae) at the N-terminus. The sequence formula of the peptoid is Ac-Nae-(Npe-Nme)4-NH2, where Ac is the acetyl group. The synthesis takes place in a commercially available solid-phase reaction vessel. The rink amide resin is used as the solid support to yield the peptoid with an amide group at the C-terminus. The resulting peptoid product is subjected to sequence analysis using a triple-quadrupole mass spectrometer coupled to an electrospray ionization source. The MS/MS measurement produces a spectrum of fragment ions resulting from the dissociation of charged peptoid. The fragment ions are sorted out based on the values of their mass-to-charge ratio (m/z). The m/z values of the fragment ions are compared against the nominal masses of theoretically predicted fragment ions, according to the scheme of peptoid fragmentation. The analysis generates a fragmentation pattern of the charged peptoid. The fragmentation pattern is correlated to the monomer sequence of the neutral peptoid. In this regard, MS analysis reads out the sequence information of the peptoids.

A protocol is described for the manual synthesis of oligo-peptoids followed by sequence analysis by mass spectrometry.

Peptoids are sequence-controlled peptide-mimicking oligomers consisting of N-alkylated glycine units. Among many potential applications, peptoids have ...

Microprobe Capillary Electrophoresis Mass Spectrometry for Single-cell Metabolomics in Live Frog (Xenopus laevis) Embryos

The quantification of small molecules in single cells raises new potentials for better understanding the basic processes that underlie embryonic development. To enable single-cell investigations directly in live embryos, new analytical approaches are needed, particularly those that are sensitive, selective, quantitative, robust, and scalable to different cell sizes. Here, we present a protocol that enables the in situ analysis of metabolism in single cells in freely developing embryos of the South African clawed frog (Xenopus laevis), a powerful model in cell and developmental biology. This approach uses a capillary microprobe to aspirate a defined portion from single identified cells in the embryo, leaving neighboring cells intact for subsequent analysis. The collected cell content is analyzed by a microscale capillary electrophoresis electrospray ionization (CE-ESI) interface coupled to a high-resolution tandem mass spectrometer. This approach is scalable to various cell sizes and compatible with the complex three-dimensional structure of the developing embryo. As an example, we demonstrate that microprobe single-cell CE-ESI-MS enables the elucidation of metabolic cell heterogeneity that unfolds as a progenitor cell gives rise to descendants during development of the embryo. Besides cell and developmental biology, the single-cell analysis protocols described here are amenable to other cell sizes, cell types, or animal models.

Microprobe Capillary Electrophoresis Mass Spectrometry for Single-cell Metabolomics in Live Frog Xenopus laevis Embryos

We describe steps that enable fast in situ sampling of a small portion of an individual cell with high precision and minimal invasion using capillary-based micro-sampling, to facilitate chemical characterization of a snapshot of metabolic activity in live embryos using a custom-built single cell capillary electrophoresis and mass spectrometry platform.

The quantification of small molecules in single cells raises new potentials for better understanding the basic processes that underlie embryonic ...

Large-scale Top-down Proteomics Using Capillary Zone Electrophoresis Tandem Mass Spectrometry

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as a useful tool for top-down proteomics that aims to characterize proteoforms in complex proteomes. However, the application of CZE-MS/MS for large-scale top-down proteomics has been impeded by the low sample-loading capacity and narrow separation window of CZE. Here, a protocol is described using CZE-MS/MS with a microliter-scale sample-loading volume and a 90-min separation window for large-scale top-down proteomics. The CZE-MS/MS platform is based on a linear polyacrylamide (LPA)-coated separation capillary with extremely low electroosmotic flow, a dynamic pH-junction-based online sample concentration method with a high efficiency for protein stacking, an electro-kinetically pumped sheath flow CE-MS interface with extremely high sensitivity, and an ion trap mass spectrometer with high mass resolution and scan speed. The platform can be used for the high-resolution characterization of simple intact protein samples and the large-scale characterization of proteoforms in various complex proteomes. As an example, a highly efficient separation of a standard protein mixture and a highly sensitive detection of many impurities using the platform is demonstrated. As another example, this platform can produce over 500 proteoform and 190 protein identifications from an Escherichia coli proteome in a single CZE-MS/MS run.

A detailed protocol is described for the separation, identification, and characterization of proteoforms in protein samples using capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS). The protocol can be used for the high-resolution characterization of proteoforms in simple protein samples and the large-scale identification of proteoforms in complex proteome samples.

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as a useful tool for top-down ...

Mass Spectrometry-Guided Genome Mining as a Tool to Uncover Novel Natural Products

The chemical space covered by natural products is immense and widely unrecognized. Therefore, convenient methodologies to perform wide-ranging evaluation of their functions in nature and potential human benefits (e.g., for drug discovery applications) are desired. This protocol describes the combination of genome mining (GM) and molecular networking (MN), two contemporary approaches that match gene cluster-encoded annotations in whole genome sequencing with chemical structure signatures from crude metabolic extracts. This is the first step towards the discovery of new natural entities. These concepts, when applied together, are defined here as MS-guided genome mining. In this method, the main components are previously designated (using MN), and structurally related new candidates are associated with genome sequence annotations (using GM). Combining GM and MN is a profitable strategy to target new molecule backbones or harvest metabolic profiles in order to identify analogues from already known compounds.

A mass spectrometry-guided genome mining protocol is established and described here. It is based on genome sequence information and LC-MS/MS analysis and aims to facilitate identification of molecules from complex microbial and plant extracts.

The chemical space covered by natural products is immense and widely unrecognized. Therefore, convenient methodologies to perform wide-ranging evaluation ...