This work was funded by the National Institute on Drug Abuse under Award no. P30DA018310 and the National Human Genome Research Institute under Award no. RM1HG010023. K.D.C. acknowledges support from a Beckman Institute Postdoctoral Fellowship. The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies.

1. Preparation of materials and solutions
<ol>
	<li>Prepare artificial seawater (ASW) with 460 mM NaCl, 10 mM KCl, 10 mM CaCl2, 22 mM MgCl2, 26 mM MgSO4, and 10 mM HEPES in water obtained from a stringent purification system. Adjust pH to 7.8 using 1 M NaOH or HCl. Typically, prepare 1 L of ASW and store at 14 &#176;C until use.</li>
	<li>Prepare an ASW-antibiotics solution with 10,000 U/mL of penicillin G, 10 &#181;g/&#181;L of streptomycin, and 10 &#181;g/&#181;L of gentamicin and store at -20 &#176;C. Immediately before the experiment, thaw and dilute the frozen antibiotics stock solution 1:100 in 20-40 mL of ASW. The final concentration of antibiotics in the working ASW solution is 100 U/mL of penicillin G, 100 &#181;g/mL of streptomycin, and 100 &#181;g/mL of gentamicin.</li>
	<li>Prepare an RNA digestion buffer by combining 1 &#181;L of 10 &#181;g/&#181;L bovine serum albumin, 0.5 &#181;L of 0.5 &#181;g/&#181;L pentostatin, 0.495 &#181;L of 2 U/&#181;L alkaline phosphatase, 1 &#181;L of 0.1 U phosphodiesterase I (in 10 mM MgCl2), and 0.38 &#181;L of endonuclease from Serratia marcescens (25 U) per sample. If analyzing multiple samples, prepare a master mix in a separate tube containing the volume of each reagent multiplied by the number of samples to be analyzed, plus one more to account for incidental reagent loss from pipet transfer steps.</li>
	<li>Prepare several sharp needles (either glass or metal) for manual isolation of neurons. Make metal needles in house by electrochemical etching of tungsten wire as in30 or purchase them. Prepare glass needles from thick or standard wall borosilicate glass capillaries (1 mm outer diameter) using a micropipette puller. For the present method, keep the needle tip diameter between 1-5 &#181;m, with a neck length of 100-150 &#181;m. 
	&#8203;NOTE: Fabrication of both metal and glass needles can be optimized to produce tools that suit the individual needs of the researcher and the specific biological model being investigated.</li>
</ol>
2. Isolation of single neuron
<ol>
	<li>Cool a 0.33 M MgCl2 solution to 14 &#176;C. Using a 50 mL syringe, anesthetize A. californica (150-250 g) by injecting the MgCl2 solution into the body cavity of the animal. Best results are obtained with 1:3 ratio of solution volume (mL) to animal body mass (g). Wait approximately 3 min for the animal to become relaxed, ensuring it does not display body contractions in response to tactile stimulation.</li>
	<li>Place the animal ventral side (foot side) up in a dissecting tray. Dissect the animal using surgical scissors with one blunt tip positioned toward the animal, carefully making a longitudinal cut through the foot.</li>
	<li>Pin the rostral, caudal, and lateral sides of the animal body to expose the internal organs and CNS ganglia located in the body cavity. Isolate major CNS ganglia from the animal by surgically severing nerves and some connectives originating from the ganglia.</li>
	<li>Immerse the ganglia in a solution of protease type XIV from Streptomyces griseus (10 mg/mL in ASW-antibiotics solution) and incubate at 34 &#176;C for 30 min or 1 h (for cerebral ganglion). 
	NOTE: The duration of incubation depends on the season, animal size and condition, as well as targeted neurons. Isolation of some neurons requires longer incubation than others and should be determined experimentally.</li>
	<li>Rinse the ganglia 6x with ASW-antibiotics solution and transfer all ganglia into in a silicone polymer-coated dish filled with ASW-antibiotics solution using a polypropylene transfer pipet that has been cut to an opening of ~5 mm. Treat the pipet with 1 mg/mL of bovine serum albumin in ASW to minimize sticking of the ganglia to the pipet (optional). Keep ganglia submerged in ASW at all times. 
	&#8203;NOTE: Enzymatic treatment reduces the mechanical stability of neurons and surrounding connective tissue and, as a result, neuronal membranes can be damaged due to exposure to air during ganglia transfer.</li>
	<li>Pin down the ganglia and use micro scissors and fine forceps to remove ganglion sheaths. With sufficiently strong enzymatic treatment, use glass or metal needles for desheathing.</li>
	<li>Visually identify A. californica neurons of interest. In this work, the following cells were studied: R2 in the abdominal ganglion, LPl1 in the pleural ganglion, metacerebral cells (MCCs) in the cerebral ganglion, and B2 cells in the buccal ganglion. Take optical images of all neural and ganglionic preparations using a calibrated microscope at 20x total magnification to determine the sizes and volumes of each cell type.</li>
	<li>Using a pulled glass capillary or sharp tungsten needles, carefully isolate the identified cell from the bulk ganglion.</li>
	<li>Draw a small amount (1 &#181;L) of ASW into a plastic micropipette, and then transfer the isolated cell into a PCR sample tube containing 4 &#181;L of 0.365 M ammonium acetate (pH 9.2). For blank measurements, collect 5 &#181;L aliquots of the ASW-antibiotics solution from the dish containing the ganglion and mix with digestion buffer (described below).</li>
</ol>
3. Cell lysis and RNA digestion for SNRMA-MS
<ol>
	<li>Lyse the isolated neurons by repeated aspiration and dispensing with a micropipette (~100 &#181;m inner diameter) in 0.365 M ammonium acetate. Some smaller cells may not immediately rupture; to lyse them, apply pressure across the diameter of the cell with a pulled glass capillary.</li>
	<li>Use a thermal cycler to heat the samples with the following temperature program: 95 &#176;C for 3 min, 10 &#176;C for 3 min, hold at 10 &#176;C. Remove the sample tube from the thermal cycler.</li>
	<li>Add 3.375 &#181;L of RNA digestion buffer for each sample and mix the solution using a micropipette by withdrawing and dispensing the solution several times. Use a miniature benchtop centrifuge at 2700 x g for 30 s to spin down any liquid droplets clinging to the walls of the PCR tube.</li>
	<li>Incubate the samples in the thermal cycler at 37 &#176;C for 3 h, followed by a hold at 10 &#176;C (heated lid set to ON). Immediately after the sample has cooled to 10 &#176;C, transfer 7 &#181;L of the solution into an autosampler vial equipped with a 250 &#181;L insert, taking care to avoid bubble formation in the autosampler tube.</li>
</ol>
4. Liquid chromatography-tandem mass spectrometry
NOTE: Analyze the single-neuron digests and authentic modified nucleoside standards using an LC-MS/MS system equipped with an electrospray ionization source and six-port diverter valve.
<ol>
	<li>To prepare the LC system for separation of canonical and modified nucleosides, equilibrate a C18 column (150 mm x 2.1 mm, 2.2 &#181;m particle size, 120 &#197; pore diameter) with 99% mobile phase A (5 mM ammonium acetate, pH 5.6) and 1% mobile phase B (60/40 mobile phase A/acetonitrile (ACN)) at a flow rate of 0.2 mL/min for 12 min at 36 &#176;C. Use LC-MS grade solvents for preparation of mobile phases.</li>
	<li>While the LC equilibrates, calibrate the mass spectrometer by introducing a 1 mM solution of sodium acetate in 50/50 ACN/water to the mass spectrometer via syringe pump with a flow rate of 5 &#181;L/min. After calibration, reconnect the LC flow to the mass spectrometer.</li>
	<li>Program the following linear gradient parameters: 1% B for 0-5 min, 5% B at 9 min, 7% B at 11 min, 10% B at 13 min, 15% B at 32 min, 40% B at 38 min, 50% B at 43 min, 100% B at 50 min, 100% B at 60 min, 1% B at 61 min, and a 12 min re-equilibration at 1% B before the next injection.</li>
	<li>Operate the MS instrument in positive mode with the following parameters: capillary voltage set to 4,500 V, drying temperature 275 &#176;C, N2 drying gas 5 L/min, and nebulizing gas 1 bar. Set the diverter valve to waste for the first 2 min of analysis and to source for the remainder of the run.</li>
	<li>Collect mass spectra over a m/z range of 110-600. Select ions for collision-induced dissociation at 35-40 eV over a 3 s cycle time using a preferred mass list constructed using the database2 and an isolation window of &#177; 0.5. Use active exclusion to exclude ions from fragmentation after three spectra.</li>
	<li>Set dynamic MS/MS spectra acquisition for ions with intensities above and below 50,000 counts at 4 Hz and 1 Hz, respectively, and a minimum threshold for ion selection at 1,990 counts.</li>
	<li>For quantitative SNRMA-MS, construct calibration curves using extracted ion chromatogram (EIC) peak areas obtained for modified nucleoside standards at a minimum of five concentrations to allow interpolation of unknown endogenous analyte concentrations. 
	&#8203;NOTE: Neurons obtained from animals with body masses of 150-250 g typically requires calibration curves for modified nucleosides ranging from 0.02 pmol to 2 pmol, but these values may vary depending on the sensitivity of the instrument.</li>
</ol>
5. Data analysis
<ol>
	<li>Generate EICs for modified nucleosides (m/z from database values2). Verify the identities of putative modified nucleosides by comparing their MS2 spectra and LC retention characteristics to database values2. See Table 1 for a list of typical RNA modifications detected in single neurons from A. californica.</li>
	<li>Manually integrate peaks that correspond to modified and canonical nucleosides and record these values in a spreadsheet. Normalize the peak area for each modified nucleoside with the sum of the peak areas for canonical cytidine, uridine, and guanosine detected in the sample. 
	NOTE: Adenosine is not included in the normalization because of its role in the CNS as a dynamic neuromodulator31.</li>
	<li>Construct a data matrix consisting of each single-neuron sample and the corresponding normalized peak areas for modified nucleosides that exhibited signal-to-noise ratios &#62;10. Perform principal component analysis (PCA) and display the first two principal components in the score plot. Construct linear calibration curves from the EIC peak areas obtained from the serial dilution of nucleoside standards and calculate the concentrations of detected analytes.</li>
</ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+quantification+of+modified+nucleosides+in+saccharomyces+cerevisiae+mRNAs.">Identification and quantification of modified nucleosides in saccharomyces cerevisiae mRNAs.</a> ACS Chemical Biology. 14 (7), 1403-1409 (2019).</li><li>Heiss, M., Reichle, V. F., Kellner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observing+the+fate+of+tRNA+and+its+modifications+by+nucleic+acid+isotope+labeling+mass+spectrometry:+NAIL-MS.">Observing the fate of tRNA and its modifications by nucleic acid isotope labeling mass spectrometry: NAIL-MS.</a> RNA Biology. 14 (9), 1260-1268 (2017).</li></ol>

The authors declare no competing financial interests.

SNRMA-MS leverages an optimized sample preparation approach, resulting in a small, MS-compatible sample volume that can be delivered to the LC-MS platform. The initial enzymatic pre-treatment of CNS ganglia dictates both the ease with which they can be desheathed and the durability of single neurons during isolation. The cerebral ganglion often requires extended enzymatic treatment due to its relatively thick sheath compared to the buccal, pleural, and abdominal ganglia. Individual researchers performing the single neuron isolations may have differing preferences for how durable the sheath should be when using microscissors and fine forceps. However, it is important that the ganglia are not over-digested, as this may lead to a loss in their structural integrity, loss of positional information that is critical for identifying target cells, and/or cell lysis. Following isolation from the ganglion, it is important to ensure that a minimal volume of isolation medium is aspirated when transferring the neuron to the sample tube. The ASW medium contains a high concentration of salts that may interfere with digestive enzymes during RNA hydrolysis and will also dilute the sample.
During the mechanical lysis step, it is common for large neurons (&#62;250 &#181;m diameter) to rupture upon repeatedly passing through a micropipette. Smaller neurons may require additional attention to ensure cell lysis, which typically involves pressing a glass capillary on the cell. In this instance, it is possible that a partial volume of the sample buffer will be drawn into the capillary because of capillary forces. This volume can be delivered back into the sample tube by applying pressure to the end of the glass capillary to ensure that no sample is lost.
The best results are obtained by including a heating step prior to adding enzymes for RNA digestion. This is likely because heating at 95 &#176;C denatures RNA secondary structures that may impede the activity of RNases35 and diminish the quantity of nucleosides released from RNA biopolymers. Control experiments using samples spiked with stable isotope-labeled methionine were previously performed to investigate whether heat-induced RNA modification artifacts were the cause of the increased peak areas observed for RNA modifications relative to a no-heat SNRMA-MS protocol29. No such labeling was observed, indicating that the improved signals for RNA modifications using the optimized SNRMA-MS method were due to superior digestion of RNA.
Conventional methods for isolating total RNA from cells involve liquid-liquid extraction (LLE) with phenol-chloroform and subsequent RNA precipitation, washing, and resuspension. These approaches have proven useful for reverse transcription polymerase chain reaction experiments where the expression of select genes can be readily monitored in identified A. californica neurons36,37. However, LLE methods cannot recover sufficient RNA for the detection of modified ribonucleosides by LC-MS, whereas the SNRMA-MS method described herein enables detection of numerous RNA modifications29. In order to assess modification profiles of specific RNA types (e.g., rRNA, tRNA, mRNA) in bulk tissue/cell samples, anion-exchange solid phase extraction24, hybridization probe-based enrichment38, and chromatographic fractionation39 approaches have been applied, but similar methods are not yet available for single-cell RNA purification. The development of RNA fractionation approaches that are capable of isolating specific RNA types from single cells would provide additional insight toward the function of RNA modifications.
SNRMA-MS revealed previously uncharacterized heterogeneity in the RNA modification landscape of single neurons in A. californica and it is conceivable that similarly distinct PTM profiles exist across mammalian cells. Because mammalian cells are relatively small compared to the large A. californica neurons analyzed in this protocol, improvements in the handling of small sample volumes are needed to facilitate lower detection limits. Though currently SNRMA-MS is limited to volumes of ~5 &#181;L, it is expected that substantial improvements can be attained by incorporating microfluidic liquid handling devices into the workflow. Furthermore, the implementation of automated or semi-automated cell isolations would increase sample throughput and permit single-cell RNA modification analysis of larger cell populations. By interfacing with nanoflow LC separations27, the characterization of epitranscriptomic marks in even smaller cells will be achievable.

Modifications in the canonical nucleosides of RNA have been increasingly recognized for their myriad roles in the regulation of protein translation. Over 150 unique RNA modifications have been reported to date that range in complexity from methylation, heteroatom addition, to conjugation with cellular metabolites1,2. This expanded RNA alphabet, also known as the epitranscriptome, is generated by enzymatic writers and is responsible for altering the stability3, folding4, and translation efficiency5,6 of cellular RNAs. Select RNA modifications may also be reversed via enzymatic erasers7,8, whereas others are appended to RNAs substoichiometrically9,10, rendering a complex landscape of modified and unmodified RNA sequences in biological systems.
The importance of RNA modifications in biological function is indicated by the unequal distribution of modifications across different organs and tissues, including subregions of the central nervous system (CNS)11. This chemical heterogeneity has been linked to CNS development12, stress response13, and activity-dependent plasticity14. The CNS subregions further comprise heterogeneous populations of cells in which individual cells exhibit distinct chemical profiles15,16,17,18. Even single cells of the same type may display unique transcriptomes, in part due to tissue microenvironments19 and stochastic gene expression20. However, while characterization of the single-cell transcriptome is somewhat routine, there are no analogous methods for establishing single-cell epitranscriptomes for multiple RNA modifications. New approaches that are capable of profiling the distribution of RNA modifications in individual cells are needed for comprehensive analysis of the cellular heterogeneity and regulatory influence of post-transcriptional modifications (PTMs) in the CNS and other biological systems.
Simultaneous measurement of numerous RNA modifications in bulk cells/tissues is readily accomplished using liquid chromatography-tandem mass spectrometry (LC-MS/MS) techniques. For LC-MS/MS analysis of modified ribonucleosides, RNA is extracted from cells (typically 103-106 cells), purified by precipitation and resuspension, and subsequently digested into nucleosides. The sample mixture consisting of canonical and modified nucleosides is then injected into the LC-MS system for analyte separation and detection, leading to determination of the full complement of RNA modifications in an organism21,22,23. LC-MS/MS was recently used to determine 26 RNA modifications in the CNS of the neurobiological model, Aplysia californica (A. californica). The abundances of some of these epitranscriptomic marks exhibited time- and region-dependent dynamics that correlated with behavioral changes in the animal24. However, it was only possible to detect RNA modifications in bulk samples containing &#62;103 cells due to the limited sensitivity of the method. These larger samples likely concealed unique and functionally important RNA modification profiles of individual cells with population averages. Although careful control of sample preparation conditions has improved the detection limits for RNA modifications in small-volume samples25,26,27,28, there remains a need for analytical methods that can detect and quantify multiple modified ribonucleosides in single cells.
This protocol introduces single-neuron RNA modification analysis by mass spectrometry (SNRMA-MS), which permits the detection of over a dozen RNA modifications in single neurons from the CNS of A. californica29. The approach consists of surgical isolation of single, identified cells from major CNS ganglia followed by an optimized sample preparation workflow involving mechanical cell lysis, RNA denaturation, and enzymatic hydrolysis in an MS-compatible buffer. Identification and quantification of post-transcriptionally modified nucleosides is then accomplished using LC-MS/MS. SNRMA-MS fulfills an unmet need in the field of RNA modification analysis by facilitating the acquisition of post-transcriptional RNA modification profiles for single neurons and has potential for future application to other cell types.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Animals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Aplysia californica</td>
			<td>National Resource for Aplysia (Miami, FL)</td>
			<td></td>
			<td>150&#8211;250 g (adult)</td>
		</tr>
		<tr>
			<td>Benchtop equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass capillary puller</td>
			<td>Sutter</td>
			<td>P-97</td>
			<td>Equipped with online degasser, autosampler, and thermostatted column compartment</td>
		</tr>
		<tr>
			<td>Milli-Q water purification system</td>
			<td>Millipore</td>
			<td></td>
			<td>Equipped with ESI source</td>
		</tr>
		<tr>
			<td>Minicentrifuge for PCR tubes</td>
			<td>LW Scientific</td>
			<td>ZS-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical Microscope</td>
			<td>Zeiss</td>
			<td>Stemi 2000C</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermocycler</td>
			<td>Techne</td>
			<td>EW-93945-01</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC column and consumables</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acclaim RSLC 120 C18 column</td>
			<td>Thermo Scientific</td>
			<td>71399</td>
			<td></td>
		</tr>
		<tr>
			<td>Autosampler vials</td>
			<td>Thermo Scientific</td>
			<td>C4011-13</td>
			<td></td>
		</tr>
		<tr>
			<td>LC and MS Instrumentation and Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DataAnalysis 4.4 software</td>
			<td>Bruker</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dionex Ultimate 3000 nanoLC</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Impact HD UHR QqTOF mass spectrometer</td>
			<td>Bruker</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RStudio</td>
			<td>RStudio</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microdissection tools</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscissors extra fine vannas 3.5&#8221;</td>
			<td>Roboz</td>
			<td>RS-5640</td>
			<td></td>
		</tr>
		<tr>
			<td>Tungsten needles</td>
			<td>Roboz</td>
			<td>RS-6065</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagents/Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4-(2-hydroxyethyl)-1-piperazineethane-sulfonic acid (HEPES)</td>
			<td>Fisher Scientific</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>Alkaline phosphatase</td>
			<td>Worthington Biochemical Corp.</td>
			<td>LS004081</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium acetate</td>
			<td>Honeywell</td>
			<td>17836</td>
			<td></td>
		</tr>
		<tr>
			<td>benzonase (endonuclease from S. marcescens)</td>
			<td>EMD Millipore</td>
			<td>70746-4</td>
			<td></td>
		</tr>
		<tr>
			<td>bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>calcium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>C4901</td>
			<td></td>
		</tr>
		<tr>
			<td>gentamycin sulfate</td>
			<td>Fisher Scientific</td>
			<td>G1264</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>M9272</td>
			<td></td>
		</tr>
		<tr>
			<td>magnesium sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>208094</td>
			<td></td>
		</tr>
		<tr>
			<td>nucleosides test mix</td>
			<td>Sigma-Aldrich</td>
			<td>47310-U</td>
			<td></td>
		</tr>
		<tr>
			<td>penicillin G</td>
			<td>Sigma-Aldrich</td>
			<td>P7794</td>
			<td></td>
		</tr>
		<tr>
			<td>pentostatin</td>
			<td>Sigma-Aldrich</td>
			<td>SML0508</td>
			<td></td>
		</tr>
		<tr>
			<td>phosphodiesterase I</td>
			<td>Worthington Biochemical Corp.</td>
			<td>LS003926</td>
			<td></td>
		</tr>
		<tr>
			<td>protease type XIV from Streptomyces griseus</td>
			<td>Sigma-Aldrich</td>
			<td>P5147</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard glass capillaries</td>
			<td>A-M Systems</td>
			<td>626000</td>
			<td>1 mm o.d., 0.5 mm i.d., 4 in</td>
		</tr>
		<tr>
			<td>streptomycin sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>S9137</td>
			<td></td>
		</tr>
	</tbody>
</table>

characterizing rna modifications in single neurons using mass spectrometry

Post-transcriptional modifications (PTMs) of RNA represent an understudied mechanism involved in the regulation of translation in the central nervous system (CNS). Recent evidence has linked specific neuronal RNA modifications to learning and memory paradigms. Unfortunately, conventional methods for the detection of these epitranscriptomic features are only capable of characterizing highly abundant RNA modifications in bulk tissues, precluding the assessment of unique PTM profiles that may exist for individual neurons within the activated behavioral circuits. In this protocol, an approach is described&#8212;single-neuron RNA modification analysis by mass spectrometry (SNRMA-MS)&#8212;to simultaneously detect and quantify numerous modified ribonucleosides in single neurons. The approach is validated using individual neurons of the marine mollusk, Aplysia californica, beginning with surgical isolation and enzymatic treatment of major CNS ganglia to expose neuron cell bodies, followed by manual single-neuron isolation using sharp needles and a micropipette. Next, mechanical and thermal treatment of the sample in a small volume of buffer is done to liberate RNA from an individual cell for subsequent RNA digestion. Modified nucleosides are then identified and quantified using an optimized liquid chromatography-mass spectrometry method. SNRMA-MS is employed to establish RNA modification patterns for single, identified neurons from A. californica that have known morphologies and functions. Examples of qualitative and quantitative SNRMA-MS are presented that highlight the heterogeneous distribution of RNA modifications across individual neurons in neuronal networks.

SNRMA-MS involves the manual isolation of identified neurons into small sample volumes for lysis, digestion, and LC-MS/MS analysis (Figure 1A). This workflow routinely detected over a dozen RNA modifications in single neurons from the CNS of A. californica (Figure 1B), representing a coverage of nearly half of the known epitranscriptome of this animal24 in a single cell. For example, subjecting the LPl1 neuron (~500 &#181;m diameter) to SNRMA-MS resulted in the detection of 15 &#177; 1 RNA modifications (n = 3). Modified nucleosides were positively identified on the basis of three attributes: LC retention properties, exact mass, and MS2 fragmentation profiles compared to values provided in the database2. Table 1 shows a list of all the RNA modifications detected in the LPl1 neuron. The high-resolution quadrupole time-of-flight mass spectrometer used for these experiments enabled a mass accuracy of 4 ppm as well as detection of the characteristic MS2 fragment ion at m/z 164 for the modified nucleoside N6,6-dimethyladenosine (m66A, Figure 1B). Combined with the LC separation data, which agrees with findings deposited in the database (m66A elutes after N6-methyladenosine (m6A)), the SNRMA-MS approach demonstrated correct assignment of modified nucleoside identities.
The SNRMA-MS platform can be leveraged for establishing RNA modification profiles of single neurons and investigating their relationship to bulk tissues. R2 neurons are large (~500 &#181;m in diameter) cholinergic cells that reside in the abdominal ganglion. SNRMA-MS was used to analyze RNA modifications in R2 neurons as well as the surrounding bulk abdominal ganglion (Figure 2A). Normalized peak areas for RNA modifications in each neuron/ganglion sample (n = 7) were used as inputs for PCA, revealing that R2 neurons exhibit distinct RNA modification profiles compared to the ganglia in which they reside (Figure 2B). This is evidenced by data points for R2 neurons and abdominal ganglia occupying different regions of the PCA score plot. Further support for unique modified nucleoside patterns was obtained from a separate cohort of animals (n = 7) in which pairwise comparisons were performed for 13 RNA modifications that were commonly detected in both the single neurons and bulk tissue (Figure 2C). Two modified nucleosides, pseudouridine (&#936;) and 2&#39;-O-methylguanosine (Gm), were at significantly higher abundance in the abdominal ganglia compared to R2 neurons. When viewing a subset of the RNA modifications with R2 neuron-ganglion pairs indicated, all of the abdominal ganglia exhibited higher levels of &#936; and Gm, and all but one of the R2 neurons had higher abundances of 2&#39;-O-methyladenosine (Am)&#160;than their corresponding ganglion (Figure 2D). Overall, the SNRMA-MS results reveal for the first time that RNA modification profiles of single cells can diverge from bulk cells in the same tissue.
Using SNRMA-MS to investigate the model animal A. californica provides a unique opportunity to characterize RNA modification profiles in identified, functionally distinct neurons. RNA modifications were evaluated by SNRMA-MS in four identified cells: R2 and LPl1 (homologous, cholinergic cells involved in defensive mucous release)32, MCCs (serotonergic modulatory neurons involved in feeding)33, and B2 cells (peptidergic neurons involved in gut motility)34. PCA of six RNA modifications in these identified neurons, either isolated immediately after enzymatic treatment or cultured in a ganglion preparation for 48 h, demonstrated the stability and dynamics of single cell epitranscriptomes. RNA modifications in functionally different cells formed unique clusters in the score plot while homologous R2/LPl1 neurons co-clustered (Figure 3A). The loading plot shows that differences were primarily driven by the abundance of positional isomers of methyladenosine, including 2&#39;-O-methyladenosine (Am) and N1-methyladenosine (m1A) (Figure 3B). In the same analysis, a comparison of freshly isolated cells and cells cultured in situ (i.e., in their respective ganglia) for 48 h was performed. As shown in the PCA score plot, functionally different cells remained distinguishable by their RNA modification profiles.
Quantitative SNRMA-MS can be used for determining absolute amounts of select RNA modifications for which authentic standards are available. External calibration curves were generated for m1A, &#936;, 2&#39;-O-methylcytidine (Cm), Am, and m6A, and the amount of each modified nucleoside in the MCC and R2/LPl1 cell pairs was determined by interpolation (Figure 4A-E). The intracellular quantities of m1A and &#936; in two pairs of symmetrical MCCs appeared to be similar, while larger differences in the amounts of these modifications were observed in three pairs of R2/LPl1 cells. In order to account for differences due to the physical size of the cells studied, RNA modification quantities were normalized by cell volumes calculated from optical measurement of cell diameters to yield intracellular concentrations of modified nucleosides. Significant differences in intracellular concentrations of Cm and Am were observed between MCCs and R2/LPl1 neurons. Overall, SNRMA-MS enables both qualitative and quantitative profiling of RNA modifications in single neurons.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63940/63940fig01.jpg" /> 
Figure 1: SNRMA-MS workflow and detection of multiple RNA modifications in single neurons by LC-MS/MS. (A) Photographs of desheathed buccal ganglion and single neuron isolation into a sample tube. Scale bar = 200 &#181;m, arrows indicate identified B1 cell. A diagram of the sample preparation procedure for LC-MS/MS analysis is also shown. (B) Overlaid EICs for RNA modifications in a single LPl1 neuron, with inset showing MS1 and MS2 spectra for N6, N6-dimethyladenosine (m66A). See Table 1 for m/z values used to generate EICs for modified nucleosides. This figure has been modified from29. <a href="https://www.jove.com/files/ftp_upload/63940/63940fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63940/63940fig02.jpg" /> 
Figure 2: SNRMA-MS distinguishes RNA modification profiles of single neurons and bulk tissue. (A) Schematic of A. californica CNS and workflow for analyzing R2 neurons and the surrounding abdominal ganglion. The photograph shows the abdominal ganglion and R2 neuron (injected with Fast Green dye for visibility). (B) Relative peak areas from 13 RNA modifications were used to generate the PCA score (top) and loading (bottom) plots. (C) Pairwise comparison of RNA modifications in the abdominal ganglion and R2 neuron. Error bars represent &#177;1 standard deviation (SD), *p &#60; 0.05, ***p &#60; 5 x 10&#8722;4, paired t-test with Bonferroni&#8722;Holm correction. (D) Comparison of select RNA modifications from panel C in which R2-abdominal ganglion pairs for each animal are shown with droplines. This figure has been modified from29. <a href="https://www.jove.com/files/ftp_upload/63940/63940fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63940/63940fig03.jpg" /> 
Figure 3: Profiling RNA modifications in identified, functionally different neurons from the A. californica CNS. (A) PCA score plot for MCCs, and B2, R2, and LPl1 cells that were either freshly isolated or isolated following in situ culture for 48 h (denoted by cMCC, cB2, cR2, cLPl1), and (B) loading plots for six RNA modifications commonly detected in these cells. This figure has been modified from29. <a href="https://www.jove.com/files/ftp_upload/63940/63940fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63940/63940fig04.jpg" /> 
Figure 4: Quantitative SNRMA-MS in A. californica neurons. Quantitative SNRMA-MS provides absolute amounts and intracellular concentrations for several modified nucleosides in single, identified A. californica neurons. Photographs of (A) left and right MCCs (LMCC and RMCC, respectively) in the cerebral ganglion and (B) R2 in the abdominal ganglion and LPl1 in the pleural ganglion. Cells are circled to enhance visibility. Linear calibration plots for (C) m1A and (D) &#936; (triangles) used for interpolation of modified nucleoside quantities in single cells (colored dots). Cell pairs from each animal are labeled 1-3. (E) Intracellular concentrations of five modified nucleosides in MCC and R2/LPl1 cell pairs. Thick lines connect cell pairs. *p &#60; 0.05, **p &#60; 0.005, paired t-test with Bonferroni&#8722;Holm correction, n = 2 animals (four cells total) for MCCs and n = 3 animals (six cells total) for R2/LPl1. This figure has been modified from29. <a href="https://www.jove.com/files/ftp_upload/63940/63940fig04large.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>RNA modification</td>
			<td>Abbreviation</td>
			<td>Elution order (C18)</td>
			<td>m/z for EIC</td>
			<td>m/z for MS2</td>
		</tr>
		<tr>
			<td>dihydrouridine</td>
			<td>D</td>
			<td>1</td>
			<td>247.09</td>
			<td>115</td>
		</tr>
		<tr>
			<td>pseudouridine</td>
			<td>Y</td>
			<td>2</td>
			<td>245.08</td>
			<td>209/179/155</td>
		</tr>
		<tr>
			<td>3-methylcytidine</td>
			<td>m3C</td>
			<td>3</td>
			<td>258.11</td>
			<td>126</td>
		</tr>
		<tr>
			<td>N1-methyladenosine</td>
			<td>m1A</td>
			<td>4</td>
			<td>282.12</td>
			<td>150</td>
		</tr>
		<tr>
			<td>5-methylcytidine</td>
			<td>m5C</td>
			<td>5</td>
			<td>258.11</td>
			<td>126</td>
		</tr>
		<tr>
			<td>N7-methylguanosine</td>
			<td>m7G</td>
			<td>6</td>
			<td>298.12</td>
			<td>166</td>
		</tr>
		<tr>
			<td>2&#39;-O-methylcytidine</td>
			<td>Cm</td>
			<td>7</td>
			<td>258.11</td>
			<td>112</td>
		</tr>
		<tr>
			<td>inosine</td>
			<td>I6A</td>
			<td>8</td>
			<td>269.09</td>
			<td>137</td>
		</tr>
		<tr>
			<td>2&#39;-O-methylguanosine</td>
			<td>Gm</td>
			<td>9</td>
			<td>298.12</td>
			<td>152</td>
		</tr>
		<tr>
			<td>N2-methylguanosine</td>
			<td>m2G</td>
			<td>10</td>
			<td>298.12</td>
			<td>166</td>
		</tr>
		<tr>
			<td>N2,N2,N7-trimethylguanosine</td>
			<td>m227G</td>
			<td>11</td>
			<td>326.15</td>
			<td>194</td>
		</tr>
		<tr>
			<td>N2,N2,-dimethylguanosine</td>
			<td>m22G</td>
			<td>12</td>
			<td>312.13</td>
			<td>180</td>
		</tr>
		<tr>
			<td>2&#39;-O-methyladenosine</td>
			<td>Am</td>
			<td>13</td>
			<td>282.12</td>
			<td>136</td>
		</tr>
		<tr>
			<td>N6-methyladenosine</td>
			<td>m6A</td>
			<td>14</td>
			<td>282.12</td>
			<td>150</td>
		</tr>
		<tr>
			<td>N6,N6-dimethyladenosine</td>
			<td>m66A</td>
			<td>15</td>
			<td>296.14</td>
			<td>164</td>
		</tr>
		<tr>
			<td>N6-isopentenyladenosine</td>
			<td>i6A</td>
			<td>16</td>
			<td>336.17</td>
			<td>204/136/148</td>
		</tr>
	</tbody>
</table>
Table 1: RNA modifications detected in single neurons from A. californica. Attributes for characterization of modified nucleosides are provided including LC retention order, m/z for generating EICs, and corresponding CID fragments.

Watch this Scientific Journal Video about Characterizing RNA Modifications in Single Neurons Using Mass Spectrometry at JoVE.com

Characterizing RNA Modifications in Single Neurons Using Mass Spectrometry

Post-transcriptional modifications of RNA represent an understudied layer of translation regulation that has recently been linked to central nervous system plasticity. Here, sample preparation and liquid chromatography-tandem mass spectrometry approach is described for simultaneous characterization of numerous RNA modifications in single neurons.

Post-transcriptional modifications (PTMs) of RNA represent an understudied mechanism involved in the regulation of translation in the central nervous ...

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2.2K Views.  University of Illinois Urbana-Champaign. Post-transcriptional modifications of RNA represent an understudied layer of translation regulation that has recently been linked to central nervous system plasticity. Here, sample preparation and liquid chromatography-tandem mass spectrometry approach is described for simultaneous characterization of numerous RNA modifications in single neurons.

Video: Characterizing RNA Modifications in Single Neurons Using Mass Spectrometry

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 ...

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 ...

Real-time Breath Analysis by Using Secondary Nanoelectrospray Ionization Coupled to High Resolution Mass Spectrometry

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental exposure in a non-invasive manner. In this work, we present a protocol to characterize the exhaled VOCs in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry (Sec-nanoESI-HRMS). The homemade Sec-nanoESI source was readily set up based on a commercial nanoESI source. Hundreds of peaks were observed in the background-subtracted mass spectra of exhaled breath, and the mass accuracy values are -4.0-13.5 ppm and -20.3-1.3 ppm in the positive and negative ion detection modes, respectively. The peaks were assigned with accurate elemental composition according to the accurate mass and isotopic pattern. Less than 30 s is used for one exhalation measurement, and it takes approximately 7 min for six replicated measurements.

A protocol for characterizing chemical composition of exhaled breath in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry is demonstrated.

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental ...

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 ...

High-throughput and Comprehensive Drug Surveillance Using Multisegment Injection-Capillary Electrophoresis-Mass Spectrometry

New analytical methods are urgently needed to enable high-throughput, yet comprehensive drug screening, given an alarming opioid and prescription drug crisis in public health. Conventional urine drug testing based on a two-tier immunoassay screen followed by a gas chromatography-tandem mass spectrometry (GC-MS/MS) or liquid chromatography-tandem mass spectrometry (LC-MS/MS) method are expensive and prone to bias while being limited to targeted panels of known drugs of abuse (DoA). Herein, we outline an improved method for drug surveillance that allows for the resolution and detection of an expanded panel of DoA and their metabolites when using multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS). Multiplexed separations of ten urine samples with a quality control by CE (&lt; 3 min/sample) in conjunction with full-scan data acquisition using a time-of-flight mass spectrometer (TOF-MS) under positive ion mode detection allows for the identification and quantification of DoA above recommended cut-off levels. An excellent resolution of drug isomers and isobars, including background interferences, are achieved when using MSI-CE-MS with an electrokinetic spacer between sample segments, where accurate mass/molecular formula together with the comigration of a matching deuterated internal standard and the detection of one or more bio-transformed metabolites facilitate DoA identification over a wider detection window. Additionally, urine samples can be analyzed directly without enzyme deconjugation for the rapid screening without complicated sample workup. MSI-CE-MS enables the surveillance of a broad spectrum of DoA that is required for the treatment monitoring of high-risk patients, including confirming prescribed drug adherence, revealing illicit drug use/substitution, and evaluating optimal dosage regimes as required for new advances in precision medicine.

Here we describe a high-throughput method for comprehensive drug surveillance that allows for the improved resolution and detection of large panels of drugs of abuse and their metabolites, with quality control based on multisegment injection-capillary electrophoresis-mass spectrometry.

New analytical methods are urgently needed to enable high-throughput, yet comprehensive drug screening, given an alarming opioid and prescription drug ...

Integrated Cell Manipulation Platform Coupled with the Single-probe for Mass Spectrometry Analysis of Drugs and Metabolites in Single Suspension Cells

Single cell mass spectrometry (SCMS) enables sensitive detection and accurate analysis of broad ranges of cellular species on the individual-cell level. The single-probe, a microscale sampling and ionization device, can be coupled with a mass spectrometer for on-line, rapid SCMS analysis of cellular constituents under ambient conditions. Previously, the single-probe SCMS technique was primarily used to measure cells immobilized onto a substrate, limiting the types of cells for studies. In the current study, the single-probe SCMS technology has been integrated with a cell manipulation system, typically used for in vitro fertilization. This integrated cell manipulation and analysis platform uses a cell-selection probe to capture identified individual floating cells and transfer the cells to the single-probe tip for microscale lysis, followed by immediate mass spectrometry analysis. This capture and transfer process removes the cells from the surrounding solution prior to analysis, minimizing the introduction of matrix molecules in the mass spectrometry analysis. This integrated setup is capable of SCMS analysis of targeted patient-isolated cells present in body fluids samples (e.g., urine, blood, saliva, etc.), allowing for potential applications of SCMS analysis to human medicine and disease biology.

An integrated cell manipulation platform is developed for use in conjunction with a single-probe mass spectrometry setup for the on-line analysis of individual suspension cells under ambient conditions.

Single cell mass spectrometry (SCMS) enables sensitive detection and accurate analysis of broad ranges of cellular species on the individual-cell level. ...

3D Depth Profile Reconstruction of Segregated Impurities Using Secondary Ion Mass Spectrometry

The presented protocol combines excellent detection limits (1 ppm to 1 ppb) using secondary ion mass spectrometry (SIMS) with reasonable spatial resolution (~1 &#181;m). Furthermore, it describes how to obtain realistic three-dimensional (3D) distributions of segregated impurities/dopants in solid state materials. Direct 3D depth profile reconstruction is often difficult to achieve due to SIMS-related measurement artifacts. Presented here is a method to identify and solve this challenge. Three major issues are discussed, including the i) nonuniformity of the detector being compensated by flat-field correction; ii) vacuum background contribution (parasitic oxygen counts from residual gases present in the analysis chamber) being estimated and subtracted; and iii)&#160;performance of all steps within a stable timespan of the primary ion source. Wet chemical etching is used to reveal the position and types of dislocation in a material, then the SIMS result is superimposed on images obtained via scanning electron microscopy (SEM). Thus, the position of agglomerated impurities can be related to the position of certain defects. The method is fast and does not require sophisticated sample preparation stage; however, it requires a high-quality, stable ion source, and the entire measurement must be performed quickly to avoid deterioration of the primary beam parameters.

The presented method describes how to identify and solve measurement artifacts related to secondary ion mass spectrometry as well as obtain realistic 3D distributions of impurities/dopants in solid state materials.