Name: spatial quantification of drugs in pulmonary tuberculosis lesions by laser capture microdissection liquid chromatography mass spectrometry (lcm-lc/ms)
Uploaded: 2018-04-18T14:00:00
Description: Watch this Scientific Journal Video about Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry LCM-LC/MS at JoVE.c

We thank Paul O&#39;Brien, Marizel Mina and Isabella Freedman for animal experiments, Jacquie Gonzalez and Danielle Weiner from NIH/NIAID for help with gamma irradiation of rabbit tissues prior to laser capture microdissection and Jansy Sarathy for manuscript thoughts and advice. This work was supported by funding from the Bill and Melinda Gates Foundation (OPP1174780) and NIH shared instrumentation grant 1S10OD018072. We thank Eliseo A. Eugenin for providing access to the Leica LMD 6500 microscope and sharing expertise and advice. The purchase, and ongoing support of, the LMD 6500 was funded by The National Institute of Mental Health grant, MH096625, the National Institute of Neurological Disorders and Stroke, NS105584, PHRI funding (to E.A.E) and GSK contributions (to E.A.E).

All animal studies were carried out in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health with approval from the Institutional Animal Care and Use Committee of the NIAID (NIH), Bethesda, MD.
1. Animal Experiments and Tissue Collection
This section of the protocol describes animal procedures and sample collection under Biosafety Level 3 (BSL3) conditions. Detailed protocols of the Mycobacterium tuberculosis</em...

<ol>
	<li>Dartois, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+path+of+anti-tuberculosis+drugs:+From+blood+to+lesions+to+mycobacterial+cells.">The path of anti-tuberculosis drugs: From blood to lesions to mycobacterial cells.</a> Nat Rev Microbiol. 12 (3), 159-167 (2014).</li><li>Prideaux, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+association+between+sterilizing+activity+and+drug+distribution+into+tuberculosis+lesions.">The association between sterilizing activity and drug distribution into tuberculosis lesions.</a> Nat Med. 21 (10), 1223-1227 (2015).</li><li>Prideaux, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-sensitivity+MALDI-MRM-MS+imaging+of+moxifloxacin+distribution+in+tuberculosis-infected+rabbit+lungs+and+granulomatous+lesions.">High-sensitivity MALDI-MRM-MS imaging of moxifloxacin distribution in tuberculosis-infected rabbit lungs and granulomatous lesions.</a> Anal Chem. 83 (6), 2112-2118 (2011).</li><li>Roscioli, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Desorption+electrospray+ionization+(DESI)+with+atmospheric+pressure+ion+mobility+spectrometry+for+drug+detection.">Desorption electrospray ionization (DESI) with atmospheric pressure ion mobility spectrometry for drug detection.</a> Analyst. 139 (7), 1740-1750 (2014).</li><li>Prideaux, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometry+imaging+of+levofloxacin+distribution+in+TB-infected+pulmonary+lesions+by+MALDI-MSI+and+continuous+liquid+microjunction+surface+sampling.">Mass spectrometry imaging of levofloxacin distribution in TB-infected pulmonary lesions by MALDI-MSI and continuous liquid microjunction surface sampling.</a> Int J Mass Spectrom. 377, 699-708 (2015).</li><li>Griffiths, R. L., Randall, E. C., Race, A. M., Bunch, J., Cooper, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Raster-mode+continuous-flow+liquid+microjunction+mass+spectrometry+imaging+of+proteins+in+thin+tissue+sections.">Raster-mode continuous-flow liquid microjunction mass spectrometry imaging of proteins in thin tissue sections.</a> Anal Chem. 89 (11), 5683-5687 (2017).</li><li>Prideaux, B., Stoeckli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometry+imaging+for+drug+distribution+studies.">Mass spectrometry imaging for drug distribution studies.</a> J Proteomics. 75 (16), 4999-5013 (2012).</li><li>Dilillo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometry+imaging,+laser+capture+microdissection,+and+LC-MS/MS+of+the+same+tissue+section.">Mass spectrometry imaging, laser capture microdissection, and LC-MS/MS of the same tissue section.</a> J Proteome Res. 16 (8), 2993-3001 (2017).</li><li>Xu, B. 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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cavitary+penetration+of+levofloxacin+among+patients+with+multidrug-resistant+tuberculosis.">Cavitary penetration of levofloxacin among patients with multidrug-resistant tuberculosis.</a> Antimicrob Agents Chemother. 59 (6), 3149-3155 (2015).</li><li>Zhao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unraveling+drug+penetration+of+echinocandin+antifungals+at+the+site+of+infection+in+an+intra-abdominal+abscess+model.">Unraveling drug penetration of echinocandin antifungals at the site of infection in an intra-abdominal abscess model.</a> Antimicrob Agents Chemother. , (2017).</li><li>Pascal, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanistic+modeling+identifies+drug-uptake+history+as+predictor+of+tumor+drug+resistance+and+nano-carrier-mediated+response.">Mechanistic modeling identifies drug-uptake history as predictor of tumor drug resistance and nano-carrier-mediated response.</a> ACS Nano. 7 (12), 11174-11182 (2013).</li></ol>

Spatially-resolved quantification of drugs within pulmonary TB lesions is required to determine whether drug exposure reaches sterilizing concentrations to bacterial populations residing within the different lesion compartments. The LCM-LC/MS method described here enables absolute quantification of anti-TB drugs within all lesion compartments, including the bacteria-rich caseum, using only 1 - 3 tissue sections in total. Traditional tissue homogenization and LC/MS approaches for drug quantification in tissue often lack t...

The ability to spatially resolve and quantify drug levels is a crucial requirement for determining whether anti-tuberculosis drugs reach bacterial subpopulations within pulmonary lesions at sterilizing concentrations1. Of particular importance is determining drug penetration into the necrotic core of the lesion (called caseum), which typically contains the highest number of bacilli and may be poorly accessible to drugs due to the absence of vascularization.
Traditional methods to assess lesion penetration, which involve homogenization of excised pulmonary lesions followed by solvent extraction and liquid chromatography mass spectrometry (LC/MS) analysis, are highly sensitive and selective for the drugs of interest. However, these methods offer poor spatial information, limited to the size of the original homogenized tissue. Mass spectrometry-based imaging approaches, such as matrix-assisted laser desorption ionization (MALDI)2,3, desorption electrospray ionization (DESI)4 or liquid-enhanced surface extraction5,6 offer highly spatially-resolved imaging capabilities, but direct quantification can be extremely challenging or impossible due to heterogeneous ion suppression effects and differing extraction efficiencies of analyte from the various cell or tissue types7. Additionally, most direct tissue MS imaging approaches are inherently less sensitive than LC/MS due to the lack of chromatographic separation of endogenous species competing for ionization and the lower solvent extraction efficiency of the drug from tissue.
Laser capture microdissection (LCM) combined with LC/MS analysis has been routinely applied to isolate and characterize distinct tissue regions for proteomic studies8,9 and recently utilized for drug quantification in dosed animal tissue10. Here we present an optimized protocol applying LCM combined with LC/MS (LCM-LC/MS) analysis to quantify anti-TB drugs within distinct granuloma compartments. In the laser capture microdissection process, a UV laser is focused through the microscope objective onto the tissue section, which cuts and isolates the desired tissue area by following a path defined by the user. For gravity-assisted LCM (the technique used for this research), the tissue section is mounted onto a thin polymer membrane slide (PET or PEN) and the tissue is captured in a collection tube cap sited below the slide. The drugs are extracted from the excised tissue and quantified using standard LC/MS approaches. The amount of tissue required to be collected is ultimately determined from the expected concentration of the drug present in the tissue and the sensitivity of the LC/MS method. For most analyses of drugs dosed at therapeutic levels and analyzed using a routine triple quadrupole mass spectrometer, 3 million &#181;m2 (3 mm2) of tissue surface area is sufficient.
This protocol describes the powerful combination of spatial profiling and full quantification offered by LCM-LC/MS, providing absolute drug concentrations within all compartments of TB granulomas. The technique may also be applied to determining drug concentrations in many different diseased tissues providing vital drug discovery and development information.

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			<td height="21" style="height:21px;width:273px;">New Zealand White rabbits</td>
			<td style="width:159px;">Covance</td>
			<td style="width:155px;">N/A</td>
			<td style="width:255px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:273px;">HN878 Mycobacterium tuberculosis</td>
			<td style="width:159px;">BEI Resources</td>
			<td style="width:155px;">NR-13647</td>
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			<td height="42" style="height:42px;width:273px;">Ketathesia (Ketamine) 100 mg/mL C3N</td>
			<td style="width:159px;">Henry Schein Animal Health</td>
			<td style="width:155px;">56344</td>
			<td></td>
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			<td height="42" style="height:42px;width:273px;">Anased (Xylazine) 100 mg/mL</td>
			<td style="width:159px;">Henry Schein Animal Health</td>
			<td style="width:155px;">33198</td>
			<td></td>
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			<td height="42" style="height:42px;width:273px;">Euthasol (pentobarbital sodium and phenytoin sodium) Solution&#160;</td>
			<td style="width:159px;">Virbac</td>
			<td style="width:155px;">710101</td>
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			<td height="21" style="height:21px;width:273px;">Acetonitrile (LC-MS grade)</td>
			<td style="width:159px;">Fisher</td>
			<td style="width:155px;">A955-212</td>
			<td></td>
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			<td height="21" style="height:21px;width:273px;">Methanol (LC-MS grade)</td>
			<td style="width:159px;">Fisher</td>
			<td style="width:155px;">A456-212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Formic Acid (LC-MS grade)</td>
			<td style="width:159px;">Fisher</td>
			<td style="width:155px;">A117-50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Water (LC-MS grade)&#160;</td>
			<td style="width:159px;">Fisher</td>
			<td style="width:155px;">W6212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">0.2 mL flat-cap PCR tubes</td>
			<td style="width:159px;">Corning</td>
			<td style="width:155px;">07-200-392</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Steel frames, PET-membrane</td>
			<td style="width:159px;">Leica</td>
			<td style="width:155px;">11505151</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Premium Frosted Microscope Slides</td>
			<td style="width:159px;">Fisher</td>
			<td style="width:155px;">12-544-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">&#160;96 Deep well plate 2.0ML PP RB&#160;</td>
			<td style="width:159px;">Fisher</td>
			<td style="width:155px;">NC0363259</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:273px;">Zorbax SB-C8&#160;column (4.6 by 50 mm; particle size, 3.5 &#956;m)</td>
			<td style="width:159px;">Agilent</td>
			<td style="width:155px;">820631-001D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">&#34;Zipper&#8221; Seal Sample Bags</td>
			<td style="width:159px;">Fisher</td>
			<td style="width:155px;">01-816-1B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Name</td>
			<td style="width:159px;">Company</td>
			<td style="width:155px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Equipment</td>
			<td style="width:159px;"></td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">CM1850&#160;cryostat</td>
			<td style="width:159px;">Leica</td>
			<td style="width:155px;">Discontinued</td>
			<td>Leica CM1860 is the current model</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Laser Microdissection System 6500</td>
			<td style="width:159px;">Leica</td>
			<td style="width:155px;">Discontinued</td>
			<td>Leica LMD 6 is the current model</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Agilent 1260 Infinity II HPLC&#160;</td>
			<td style="width:159px;">Agilent</td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">API 4000 QTRAP Mass Spectrometer</td>
			<td style="width:159px;">Sciex</td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

spatial quantification of drugs in pulmonary tuberculosis lesions by laser capture microdissection liquid chromatography mass spectrometry (lcm-lc/ms)

Tuberculosis is still a leading cause of morbidity and mortality worldwide. Improvements to existing drug regimens and the development of novel therapeutics are urgently required. The ability of dosed TB drugs to reach and sterilize bacteria within poorly-vascularized necrotic regions (caseum) of pulmonary granulomas is crucial for successful therapeutic intervention. Effective therapeutic regimens must therefore contain drugs with favorable caseum penetration properties. Current LC/MS methods for quantifying drug levels in biological tissues have limited spatial resolution capabilities, making it difficult to accurately determine absolute drug concentrations within small tissue compartments such as those found within necrotic granulomas. Here we present a protocol combining laser capture microdissection (LCM) of pathologically-distinct tissue regions with LC/MS quantification. This technique provides absolute quantification of drugs within granuloma caseum, surrounding cellular lesion and uninvolved lung tissue and, therefore, accurately determines whether bactericidal concentrations are being achieved. In addition to tuberculosis research, the technique has many potential applications for spatially-resolved quantification of drugs in diseased tissues.

An overview of the LCM-LC/MS approach is shown in Figure 1. After sterilizing the tissue by gamma-irradiation, all subsequent steps (from tissue sectioning onwards) take place outside of BSL3 conditions. Figure 2 shows the lesion biopsy sections before and after tissue isolation by LCM. Necrotic and cellular areas of TB lesions can be easily identified and isolated by visual inspection of optical images alone (without the require...

Watch this Scientific Journal Video about Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry LCM-LC/MS at JoVE.c

Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry LCM-LC/MS

Here, we describe a protocol using laser capture microdissection coupled with LC/MS analysis to spatially-quantify drug distributions within pulmonary tuberculosis granulomas. The approach has broad applicability to quantifying drug concentrations within tissues at high spatial detail.

Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry (LCM-LC/MS)

Tuberculosis is still a leading cause of morbidity and mortality worldwide. Improvements to existing drug regimens and the development of novel ...

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Unit 1

Lower Respiratory Tract Infections

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Magnetic Resonance Imaging Quantification of Pulmonary Perfusion using Calibrated Arterial Spin Labeling

This demonstrates a MR imaging method to measure the spatial distribution of pulmonary blood flow in healthy subjects 
during normoxia (inspired O2, fraction (FIO2) = 0.21) hypoxia (FIO2 = 0.125), and hyperoxia 
(FIO2 = 1.00). In addition, the physiological responses of the subject are monitored in the MR scan environment. MR images 
were obtained on a 1.5 T GE MRI scanner during a breath hold from a sagittal slice in the right lung at functional residual capacity. An arterial 
spin labeling sequence (ASL-FAIRER) was used to measure the spatial distribution of pulmonary blood flow 1,2 and a multi-echo fast 
gradient echo (mGRE) sequence 3 was used to quantify the regional proton (i.e. H2O) density, allowing the quantification 
of density-normalized perfusion for each voxel (milliliters blood per minute per gram lung tissue). 
With a pneumatic switching valve and facemask equipped with a 2-way non-rebreathing valve, different oxygen concentrations 
were introduced to the subject in the MR scanner through the inspired gas tubing. A metabolic cart collected expiratory gas via expiratory tubing. Mixed expiratory O2 and CO2 concentrations, oxygen consumption, carbon dioxide production, respiratory exchange ratio, 
respiratory frequency and tidal volume were measured. Heart rate and oxygen saturation were monitored using pulse-oximetry. 
Data obtained from a normal subject showed that, as expected, heart rate was higher in hypoxia (60 bpm) than during normoxia (51) or hyperoxia (50) and the arterial oxygen saturation (SpO2) was reduced during hypoxia to 86%. Mean ventilation was 8.31 L/min BTPS during hypoxia, 7.04 L/min during normoxia, and 6.64 L/min during hyperoxia. Tidal volume was 0.76 L during hypoxia, 0.69 L during normoxia, and 0.67 L during hyperoxia. 
Representative quantified ASL data showed that the mean density normalized perfusion was 8.86 ml/min/g during hypoxia, 8.26 ml/min/g during normoxia and 8.46 ml/min/g during hyperoxia, respectively. In this subject, the relative dispersion4, an index of global heterogeneity, was increased in hypoxia (1.07 during hypoxia, 0.85 during normoxia, and 0.87 during hyperoxia) while the fractal dimension (Ds), another index of heterogeneity reflecting vascular branching structure, was unchanged (1.24 during hypoxia, 1.26 during normoxia, and 1.26 during hyperoxia). 
Overview. This protocol will demonstrate the acquisition of data to measure the distribution of pulmonary perfusion noninvasively under conditions of normoxia, hypoxia, and hyperoxia using a magnetic resonance imaging technique known as arterial spin labeling (ASL). 
Rationale: Measurement of pulmonary blood flow and lung proton density using MR technique offers high spatial resolution images which can be quantified and the ability to perform repeated measurements under several different physiological conditions. In human studies, PET, SPECT, and CT are commonly used as the alternative techniques. However, these techniques involve exposure to ionizing radiation, and thus are not suitable for repeated measurements in human subjects.

A MR imaging method to study the distribution of pulmonary blood flow under a variety of physiological conditions, in this case exposure to three different inspired oxygen concentrations: hypoxia, normoxia, and hyperoxia, is described. This technique utilizes human pulmonary physiology research techniques in an MR scanning environment.

This demonstrates a MR imaging method to measure the spatial distribution of pulmonary blood flow in healthy subjects 
during normoxia (inspired O2, ...

MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson's Disease

MALDI imaging mass spectrometry (IMS) is a powerful approach that facilitates the spatial analysis of molecular species in biological tissue samples2 (Fig.1). A 12 &mu;m thin tissue section is covered with a MALDI matrix, which facilitates desorption and ionization of intact peptides and proteins that can be detected with a mass analyzer, typically using a MALDI TOF/TOF mass spectrometer. Generally hundreds of peaks can be assessed in a single rat brain tissue section. In contrast to commonly used imaging techniques, this approach does not require prior knowledge of the molecules of interest and allows for unsupervised and comprehensive analysis of multiple molecular species while maintaining high molecular specificity and sensitivity2. Here we describe a MALDI IMS based approach for elucidating region-specific distribution profiles of neuropeptides in the rat brain of an animal model Parkinson's disease (PD). 
PD is a common neurodegenerative disease with a prevalence of 1% for people over 65 of age3,4. The most common symptomatic treatment is based on dopamine replacement using L-DOPA5. However this is accompanied by severe side effects including involuntary abnormal movements, termed L-DOPA-induced dyskinesias (LID)1,3,6. One of the most prominent molecular change in LID is an upregulation of the opioid precursor prodynorphin mRNA7. The dynorphin peptides modulate neurotransmission in brain areas that are essentially involved in movement control7,8. However, to date the exact opioid peptides that originate from processing of the neuropeptide precursor have not been characterized. Therefore, we utilized MALDI IMS in an animal model of experimental Parkinson's disease and L-DOPA induced dyskinesia. 
MALDI imaging mass spectrometry proved to be particularly advantageous with respect to neuropeptide characterization, since commonly used antibody based approaches targets known peptide sequences and previously observed post-translational modifications. By contrast MALDI IMS can unravel novel peptide processing products and thus reveal new molecular mechanisms of neuropeptide modulation of neuronal transmission. While the absolute amount of neuropeptides cannot be determined by MALDI IMS, the relative abundance of peptide ions can be delineated from the mass spectra, giving insights about changing levels in health and disease. In the examples presented here, the peak intensities of dynorphin B, alpha-neoendorphin and substance P were found to be significantly increased in the dorsolateral, but not the dorsomedial, striatum of animals with severe dyskinesia involving facial, trunk and orolingual muscles (Fig. 5). Furthermore, MALDI IMS revealed a correlation between dyskinesia severity and levels of des-tyrosine alpha-neoendorphin, representing a previously unknown mechanism of functional inactivation of dynorphins in the striatum as the removal of N-terminal tyrosine reduces the dynorphin's opioid-receptor binding capacity9. This is the first study on neuropeptide characterization in LID using MALDI IMS and the results highlight the potential of the technique for application in all fields of biomedical research.

Dopamine replacement pharmacotherapy using L-DOPA is the most commonly used symptomatic treatment of Parkinson&#x2019;s disease, but is accompanied by side effects including involuntary abnormal movements, termed dyskinesia 1. Here, a protocol for MALDI imaging mass spectrometry is presented that detects changes in rat brain neuropeptide levels related to dyskinesia.

MALDI imaging mass spectrometry (IMS) is a powerful approach that facilitates the spatial analysis of molecular species in biological tissue samples2 ...

Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected cells can be used for a variety of investigations, such as transcriptomic or proteomic studies, DNA assessment or chromosomal analysis 2,3. An especially challenging application of LMD is transcriptome analysis, which, due to the lability of RNA 4, can be particularly prominent when cells are dissected from tissues that are rich of RNases, such as the pancreas. A microdissection protocol that enables fast identification and collection of target cells is essential in this setting in order to shorten the tissue handling time and, consequently, to ensure RNA preservation. 
Here we describe a protocol for acquiring human pancreatic beta cells from surgical specimens to be used for transcriptomic studies 5. Small pieces of pancreas of about 0.5-1 cm3 were cut from the healthy appearing margins of resected pancreas specimens, embedded in Tissue-Tek O.C.T. Compound, immediately frozen in chilled 2-Methylbutane, and stored at -80 &deg;C until sectioning. Forty serial sections of 10 &mu;m thickness were cut on a cryostat under a -20 &deg;C setting, transferred individually to glass slides, dried inside the cryostat for 1-2 min, and stored at -80 &deg;C. 
Immediately before the laser microdissection procedure, sections were fixed in ice cold, freshly prepared 70% ethanol for 30 sec, washed by 5-6 dips in ice cold DEPC-treated water, and dehydrated by two one-minute incubations in ice cold 100% ethanol followed by xylene (which is used for tissue dehydration) for 4 min; tissue sections were then air-dried afterwards for 3-5 min. Importantly, all steps, except the incubation in xylene, were performed using ice-cold reagents - a modification over a previously described protocol 6. utilization of ice cold reagents resulted in a pronounced increase of the intrinsic autofluorescence of beta cells, and facilitated their recognition. For microdissection, four sections were dehydrated each time: two were placed into a foil-wrapped 50 ml tube, to protect the tissue from moisture and bleaching; the remaining two were immediately microdissected. This procedure was performed using a PALM MicroBeam instrument (Zeiss) employing the Auto Laser Pressure Catapulting (AutoLPC) mode. The completion of beta cell/islet dissection from four cryosections required no longer than 40-60 min. Cells were collected into one AdhesiveCap and lysed with 10 &mu;l lysis buffer. Each single RNA specimen for transcriptomic analysis was obtained by combining 10 cell microdissected samples, followed by RNA extraction using the Pico Pure RNA Isolation Kit (Arcturus). This protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their rapid and accurate recognition and collection. Further improvement of this procedure could enable the dissection of phenotypically different beta cells, with possible implications for better understanding the changes associated with type 2 diabetes.

Laser microdissection is a technique that allows the recovery of selected cells from minute amounts of parenchyma. Here we describe a protocol for acquiring human pancreatic islets from surgical specimens to be used for transcriptomic studies. Our protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their collection.

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected ...

Quantitative Mass Spectrometric Profiling of Cancer-cell Proteomes Derived From Liquid and Solid Tumors

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic biomarkers. However, methods for quantitative proteomic characterization of patient-derived tumors and in particular their cellular subpopulations are largely lacking. Here we describe an experimental set-up that allows quantitative analysis of proteomes of cancer cell subpopulations derived from either liquid or solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry followed by bioinformatic data analysis. To enrich specific cellular subsets, liquid tumors are first immunophenotyped by flow cytometry followed by FACS-sorting; for solid tumors, laser-capture microdissection is used to purify specific cellular subpopulations. In a second step, proteins are extracted from the purified cells and subsequently combined with a tumor-specific, SILAC-labeled spike-in standard that enables protein quantification. The resulting protein mixture is subjected to either gel electrophoresis or Filter Aided Sample Preparation (FASP) followed by tryptic digestion. Finally, tryptic peptides are analyzed using a hybrid quadrupole-orbitrap mass spectrometer, and the data obtained are processed with bioinformatic software suites including MaxQuant. By means of the workflow presented here, up to 8,000 proteins can be identified and quantified in patient-derived samples, and the resulting protein expression profiles can be compared among patients to identify diagnostic proteomic signatures or potential drug targets.

In-depth analyses of cancer cell proteomes facilitate identification of novel drug targets and diagnostic biomarkers. We describe an experimental workflow for quantitative analysis of (phospho-)proteomes in cancer cell subpopulations derived from liquid and solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry.

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic ...

Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis

The prostate gland contains a heterogeneous milieu of stromal, epithelial, neuroendocrine and immune cell types. Healthy prostate is comprised of fibromuscular stroma surrounding discrete epithelial-lined secretory lumens and a very small population of immune and neuroendocrine cells. In contrast, areas of prostate cancer have increased dysplastic luminal epithelium with greatly reduced or absent stromal population. Given the profound difference between stromal and epithelial cell types, it is imperative to separate the cell types for any type of downstream molecular analysis. Despite this knowledge, the bulk of gene expression studies compare benign prostate to cancer without micro-dissection, leading to stromal bias in the benign samples. Laser-capture micro-dissection (LCM) is an effective method to physically separate different cell types from a specimen section. The goal of this protocol is to show that RNA can be successfully isolated from LCM-collected human prostatic epithelium and used for downstream gene expression studies such as RT-qPCR and RNAseq.

The goal of this protocol is to use laser-capture micro-dissection as an effective method to isolate pure populations of cell types from heterogeneous prostate tissues for downstream RNA analysis.

The prostate gland contains a heterogeneous milieu of stromal, epithelial, neuroendocrine and immune cell types.  Healthy prostate is comprised of ...

Imaging Metals in Brain Tissue by Laser Ablation - Inductively Coupled Plasma - Mass Spectrometry (LA-ICP-MS)

Metals are found ubiquitously throughout an organism, with their biological role dictated by both their chemical reactivity and abundance within a specific anatomical region. Within the brain, metals have a highly compartmentalized distribution, depending on the primary function they play within the central nervous system. Imaging the spatial distribution of metals has provided unique insight into the biochemical architecture of the brain, allowing direct correlation between neuroanatomical regions and their known function with regard to metal-dependent processes. In addition, several age-related neurological disorders feature disrupted metal homeostasis, which is often confined to small regions of the brain that are otherwise difficult to analyze. Here, we describe a comprehensive method for quantitatively imaging metals in the mouse brain, using laser ablation - inductively coupled plasma - mass spectrometry (LA-ICP-MS) and specially designed image processing software. Focusing on iron, copper and zinc, which are three of the most abundant and disease-relevant metals within the brain, we describe the essential steps in sample preparation, analysis, quantitative measurements and image processing to produce maps of metal distribution within the low micrometer resolution range. This technique, applicable to any cut tissue section, is capable of demonstrating the highly variable distribution of metals within an organ or system, and can be used to identify changes in metal homeostasis and absolute levels within fine anatomical structures.

Imaging Metals in Brain Tissue by Laser Ablation - Inductively Coupled Plasma - Mass Spectrometry LA-ICP-MS

Quantitatively mapping metals in tissue by laser ablation - inductively coupled plasma - mass spectrometry (LA-ICP-MS) is a sensitive analytical technique that can provide new insight into how metals participate in normal function and disease processes. Here, we describe a protocol for quantitatively imaging metals in thin sections of mouse neurological tissue.

Metals are found ubiquitously throughout an organism, with their biological role dictated by both their chemical reactivity and abundance within a ...

An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products

The gut-blood barrier (GBB) controls the passage of nutrients, bacterial metabolites and drugs from intestinal lumen to the bloodstream. The GBB integrity is disturbed in gastrointestinal, cardiovascular and metabolic diseases, which may result in easier access of biologically active compounds, such as gut bacterial metabolites, to the bloodstream. Thus, the permeability of the GBB may be a marker of both intestinal and extraintestinal diseases. Furthermore, the increased penetration of bacterial metabolites may affect the functioning of the entire organism.
Commonly used methods for studying the GBB permeability are performed ex vivo. The accuracy of those methods is limited, because the functioning of the GBB depends on intestinal blood flow. On the other hand, commonly used in vivo methods may be biased by liver and kidney performance, as those methods are based on evaluation of urine or/and peripheral blood concentrations of exogenous markers. Here, we present a direct measurement of GBB permeability in rats using an in vivo method based on portal blood sampling, which preserves intestinal blood flow and is virtually not affected by the liver and kidney function. 
Polyurethane catheters are inserted into the portal vein and inferior vena cava just above the hepatic veins confluence. Blood is sampled at baseline and after administration of a selected marker into a desired part of the gastrointestinal tract. Here, we present several applications of the method including (1) evaluation of the colon permeability to TMA, a gut bacterial metabolite, (2) evaluation of liver clearance of TMA, and (3) evaluation of a gut-portal blood-liver-peripheral blood pathway of gut bacteria-derived short-chain fatty acids. Furthermore, the protocol may also be used for tracking intestinal absorption and liver metabolism of drugs or for measurements of portal blood pressure.

An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products

The access of nutrients, microbiota metabolites and medicines to the circulation is controlled by the gut-blood barrier (GBB). We describe a direct method for measuring the GBB permeability in vivo, which, in contrast to commonly used indirect methods, is virtually not affected by liver and kidney functions.

The gut-blood barrier (GBB) controls the passage of nutrients, bacterial metabolites and drugs from intestinal lumen to the bloodstream. The GBB integrity ...

Immunoglobulin G N-Glycan Analysis by Ultra-Performance Liquid Chromatography

Glycomics is a new subspecialty in omics system research that offers significant potential in discovering next-generation biomarkers for disease susceptibility, drug target discovery, and precision medicine. Alternative IgG N-glycans have been reported in several common chronic diseases and suggested to have great potential in clinical applications (i.e., biomarkers for diagnosis and prediction of diseases). IgG N-glycans are widely characterized using the method of hydrophilic interaction chromatography (HILIC) ultra-performance liquid chromatography (UPLC). UPLC is a stable detection technology with good reproducibility and high relative quantitative accuracy. In addition, the structure of IgG N-glycan is clearly separated, and glycan composition and relative abundance in plasma are characterized.

Immunoglobulin G (IgG) N-glycan is characterized using hydrophilic interaction chromatography UPLC. In addition, the structure of IgG N-glycan is clearly separated. Presented here is an introduction to this experimental method so that it can be widely used in research settings.

Glycomics is a new subspecialty in omics system research that offers significant potential in discovering next-generation biomarkers for disease ...

Analysis of Raw and Processed Cyperi Rhizoma Samples Using Liquid Chromatography-Tandem Mass Spectrometry in Rats with Primary Dysmenorrhea

Cyperi rhizoma (CR) is widely used in gynecology and is a general medicine for treating women's diseases in China. Since the analgesic effect of CR is enhanced after processing with vinegar, CR processed with vinegar (CRV) is generally used clinically. However, the mechanism by which the analgesic effect is enhanced by vinegar processing is unclear. In this study, the ultra-high pressure liquid chromatographytandem mass spectrometry (UPLC-MS/MS) technique was used to examine changes in the blood levels of the exogenous constituents and metabolites between CR-treated and CRV-treated rats with dysmenorrhea. The results revealed differing levels of 15 constituents and two metabolites in the blood of these rats. Among them, the levels of (-)-myrtenol and [(1R,2S,3R,4R)-3-hydroxy-1,4,7,7-tetramethylbicyclo[2.2.1]hept-2-yl]acetic acid in the CRV group were considerably higher than in the CR group. CRV reduced the level of 2-series prostanoids and 4-series leukotrienes with proinflammatory, platelet aggregation, and vasoconstriction activities and provided analgesic effects by modulating arachidonic acid and linoleic acid metabolism and the biosynthesis of unsaturated fatty acids. This study revealed that vinegar processing enhances the analgesic effect of CR and contributes to our understanding of the mechanism of action of CRV.

Here, a comparative analysis of raw and processed Cyperi rhizoma (CR) samples is presented using ultra-high performance liquid chromatography-high-resolution tandem mass spectrometry (UPLC-MS/MS) in rats with primary dysmenorrhea. The changes in blood levels of the metabolites and the sample constituents were examined between rats treated with CR and CR processed with vinegar (CRV).

Cyperi rhizoma (CR) is widely used in gynecology and is a general medicine for treating women's diseases in China. Since the analgesic effect of CR is ...

Capturing Oral Submucous Fibrosis Tissue Samples for Spatial-Omics Analysis

Isolation of Cells with Morphological and Spatial Information from Oral Submucous Fibrosis Samples by Laser Capture Microdissection

Oral submucous fibrosis (OSF) is a common type of potentially malignant disorder in the oral cavity. The atrophy of epithelium and fibrosis of the lamina propria and the submucosa are often found on histopathological slides. Epithelial dysplasia, epithelial atrophy, and senescent fibroblasts have been proposed to be associated with the malignant transformation of OSF. However, because of the heterogeneity of potentially malignant oral disorders and oral squamous cell carcinoma, it is difficult to identify the specific molecular mechanisms of malignant transformation in OSF. Here, we present a method to obtain a small number of epithelial or mesenchymal cells carrying morphological data and spatial information by laser capture microdissection on formalin-fixed paraffin-embedded tissue slides. Using a microscope, we can precisely capture microscale (~500 cells) dysplastic or atrophic epithelial tissue and fibrotic subepithelial tissue. The extracted cells can be evaluated by genome or transcriptome sequencing to acquire genomic and transcriptomic data with morphological and spatial information. This approach removes the heterogeneity of bulk OSF tissue sequencing and the interference caused by cells in non-lesioned areas, allowing for precise spatial-omics analysis of OSF tissue.

Author Spotlight: Unlocking the Mysteries of Oral Potential Malignancies 

Laser capture microdissection of oral submucous fibrosis tissues allows for precise extraction of cells from histological regions of interest for the analysis of multi-omics data with morphological and spatial information.

Oral submucous fibrosis (OSF) is a common type of potentially malignant disorder in the oral cavity. The atrophy of epithelium and fibrosis of the lamina ...