Name: whole-body mass spectrometry imaging by infrared matrix-assisted laser desorption electrospray ionization (ir-maldesi)
Uploaded: 2016-03-24T15:00:00
Description: Watch this Scientific Journal Video about Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization IR-MALDESI at JoVE.com

The authors thank Professor H. Troy Ghashghaei from NCSU Department of Molecular Biomedical Sciences for providing the whole mouse tissue. The authors also gratefully acknowledge the financial assistance received from National Institutes of Health (R01GM087964), the W.M. Keck foundation, and North Carolina State University.

Note: The following protocol describes all the necessary steps for performing IR-MALDESI MSI experiments. In-depth details about the optimized geometry of the IR-MALDESI source and its synchronization with the laser, stage, and mass spectrometer can be found elsewhere5,6. Animal tissue samples used in this protocol were obtained according to Institutional Animal Care and Use Committee (IACUC) and North Carolina State University regulations.
1. Tissue Preparation
<ol>
	<li>Prepare ...

<ol>
	<li>Mcdonnell, L. A., Heeren, R. M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Mass+Spectrometry.">Imaging Mass Spectrometry.</a> Mass Spectrom. Rev. 26, 606-643 (2007).</li><li>Chughtai, K., Heeren, R. M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometric+imaging+for+biomedical+tissue+analysis.">Mass spectrometric imaging for biomedical tissue analysis.</a> Chem. Rev. 110 (5), 3237-3277 (2010).</li><li>Robichaud, G., Barry, J. A., Muddiman, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atmospheric+Pressure+Mass+Spectrometry+Imaging.">Atmospheric Pressure Mass Spectrometry Imaging.</a> Encycl. Anal. Chem. , (2014).</li><li>Sampson, J. S., Hawkridge, A. M., Muddiman, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+and+detection+of+multiply-charged+peptides+and+proteins+by+matrix-assisted+laser+desorption+electrospray+ionization+(MALDESI)+Fourier+transform+ion+cyclotron+resonance+mass+spectrometry.">Generation and detection of multiply-charged peptides and proteins by matrix-assisted laser desorption electrospray ionization (MALDESI) Fourier transform ion cyclotron resonance mass spectrometry.</a> J. Am. Soc. Mass Spectrom. 17 (12), 1712-1716 (2006).</li><li>Robichaud, G., Barry, J. A., Garrard, K. P., Muddiman, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infrared+matrix-assisted+laser+desorption+electrospray+ionization+(IR-MALDESI)+imaging+source+coupled+to+a+FT-ICR+mass+spectrometer.">Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) imaging source coupled to a FT-ICR mass spectrometer.</a> J. Am. Soc. Mass Spectrom. 24 (1), 92-100 (2013).</li><li>Robichaud, G., Barry, J. A., Muddiman, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IR-MALDESI+Mass+Spectrometry+Imaging+of+Biological+Tissue+Sections+Using+Ice+as+a+Matrix.">IR-MALDESI Mass Spectrometry Imaging of Biological Tissue Sections Using Ice as a Matrix.</a> J. Am. Soc. Mass Spectrom. 25 (3), 319-328 (2014).</li><li>Barry, J. A., 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=Mapping+Antiretroviral+Drugs+in+Tissue+by+IR-MALDESI+MSI+Coupled+to+the+Q+Exactive+and+Comparison+with+LC-MS/MS+SRM+Assay.">Mapping Antiretroviral Drugs in Tissue by IR-MALDESI MSI Coupled to the Q Exactive and Comparison with LC-MS/MS SRM Assay.</a> J. Am. Soc. Mass Spectrom. 25 (12), 2038-2047 (2014).</li><li>Rosen, E. P., Bokhart, M. T., Ghashghaei, H. T., Muddiman, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+Desorption+Conditions+on+Analyte+Sensitivity+and+Internal+Energy+in+Discrete+Tissue+or+Whole+Body+Imaging+by+IR-MALDESI.">Influence of Desorption Conditions on Analyte Sensitivity and Internal Energy in Discrete Tissue or Whole Body Imaging by IR-MALDESI.</a> J. Am. Soc. Mass Spectrom. 26, 899-910 (2015).</li><li>Nelson, K. A., Daniels, G. J., Fournie, J. W., Hemmer, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+whole-body+zebrafish+sectioning+methods+for+mass+spectrometry+imaging.">Optimization of whole-body zebrafish sectioning methods for mass spectrometry imaging.</a> J. Biomol. Tech. 24 (3), 119-127 (2013).</li><li>Park, J. J., Cunningham, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thin+sectioning+of+slice+preparations+for+immunohistochemistry.">Thin sectioning of slice preparations for immunohistochemistry.</a> J. Vis. Exp. (3), e194 (2007).</li><li>Bokhart, M. T., Rosen, E., Thompson, C., Sykes, C., Kashuba, A. D. M., Muddiman, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+mass+spectrometry+imaging+of+emtricitabine+in+cervical+tissue+model+using+infrared+matrix-assisted+laser+desorption+electrospray+ionization.">Quantitative mass spectrometry imaging of emtricitabine in cervical tissue model using infrared matrix-assisted laser desorption electrospray ionization.</a> Anal. Bioanal. Chem. 407 (8), 2073-2084 (2015).</li><li>Nazari, M., Muddiman, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarity+Switching+Mass+Spectrometry+Imaging+of+Healthy+and+Cancerous+Hen+Ovarian+Tissue+Sections+by+Infrared+Matrix-Assisted+Laser+Desorption+Electrospray+Ionization+(IR-MALDESI).">Polarity Switching Mass Spectrometry Imaging of Healthy and Cancerous Hen Ovarian Tissue Sections by Infrared Matrix-Assisted Laser Desorption Electrospray Ionization (IR-MALDESI).</a> Analyst. 141, 595-605 (2016).</li><li>Hsu, C. C., 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=Design+and+Application+of+a+Low-Temperature+Peltier-Cooling+Microscope.">Design and Application of a Low-Temperature Peltier-Cooling Microscope.</a> J. Pharm. Sci. 85 (1), 70-74 (1996).</li><li>Jurchen, J. C., Rubakhin, S. S., Sweedler, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MALDI-MS+imaging+of+features+smaller+than+the+size+of+the+laser+beam.">MALDI-MS imaging of features smaller than the size of the laser beam.</a> J. Am. Soc.Mass Spectrom. 16 (10), 1654-1659 (2005).</li><li>Nazari, M., Muddiman, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular-level+mass+spectrometry+imaging+using+infrared+matrix-assisted+laser+desorption+electrospray+ionization+(IR-MALDESI)+by+oversampling.">Cellular-level mass spectrometry imaging using infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) by oversampling.</a> Anal. Bioanal. Chem. 407 (8), 2265-2271 (2015).</li><li>Rosen, E. P., Bokhart, M. T., Nazari, M., Muddiman, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+C-Trap+Ion+Accumulation+Time+on+the+Detectability+of+Analytes+in+IR-MALDESI+MSI.">Influence of C-Trap Ion Accumulation Time on the Detectability of Analytes in IR-MALDESI MSI.</a> Anal. Chem. 87, 10483-10490 (2015).</li><li>Kessner, D., Chambers, M., Burke, R., Agus, D., Mallick, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ProteoWizard:+open+source+software+for+rapid+proteomics+tools+development.">ProteoWizard: open source software for rapid proteomics tools development.</a> Bioinformatics. 24 (21), 2534-2536 (2008).</li><li>Schramm, T., 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=ImzML+-+A+common+data+format+for+the+flexible+exchange+and+processing+of+mass+spectrometry+imaging+data.">ImzML - A common data format for the flexible exchange and processing of mass spectrometry imaging data.</a> J. Proteomics. 75 (16), 5106-5110 (2012).</li><li>Race, A. M., Styles, I. B., Bunch, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inclusive+sharing+of+mass+spectrometry+imaging+data+requires+a+converter+for+all.">Inclusive sharing of mass spectrometry imaging data requires a converter for all.</a> J. Proteomics. 75 (16), 5111-5112 (2012).</li><li>Robichaud, G., Garrard, K. P., Barry, J. A., Muddiman, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MSiReader:+an+open-source+interface+to+view+and+analyze+high+resolving+power+MS+imaging+files+on+Matlab+platform.">MSiReader: an open-source interface to view and analyze high resolving power MS imaging files on Matlab platform.</a> J. Am. Soc. Mass Spectrom. 24 (5), 718-721 (2013).</li><li>Smith, C. A., O'Maille, G., 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=METLIN:+a+metabolite+mass+spectral+database.">METLIN: a metabolite mass spectral database.</a> Ther. Drug. Monit. 27 (6), 747-751 (2005).</li><li>Sud, 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=LMSD:+LIPID+MAPS+structure+database.">LMSD: LIPID MAPS structure database.</a> Nucleic Acids Res. 35, D527-D532 (2007).</li><li>Schwartz, S. A., Reyzer, M. L., Caprioli, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+tissue+analysis+using+matrix-assisted+laser+desorption/ionization+mass+spectrometry:+practical+aspects+of+sample+preparation.">Direct tissue analysis using matrix-assisted laser desorption/ionization mass spectrometry: practical aspects of sample preparation.</a> J. Mass Spectrom. 38 (7), 699-708 (2003).</li><li>Takai, N., Tanaka, Y., Inazawa, K., Saji, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+pharmaceutical+drug+distribution+in+multiple+organs+by+imaging+mass+spectrometry.">Quantitative analysis of pharmaceutical drug distribution in multiple organs by imaging mass spectrometry.</a> Rapid Commun. Mass Spectrom. 26 (13), 1549-1556 (2012).</li><li>Liu, J., Gingras, J., Ganley, K. P., Vismeh, R., Teffera, Y., Zhao, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-body+tissue+distribution+study+of+drugs+in+neonate+mice+using+desorption+electrospray+ionization+mass+spectrometry+imaging.">Whole-body tissue distribution study of drugs in neonate mice using desorption electrospray ionization mass spectrometry imaging.</a> Rapid Commun. Mass Spectrom. 28 (2), 185-190 (2014).</li></ol>

The protocol above describes the key steps for performing an IR-MALDESI MSI experiment. The matrix application process (Section 3) takes approximately 20 min, which is similar to a typical matrix application process for MALDI MSI experiments by sublimation or spray-coating using a robotic sprayer. Furthermore, IR-MALDESI does not rely on partitioning of analytes into the matrix crystals6, and the ice matrix can be universally used for all analytes regardless of their mass, size, or chemical properties. In addi...

Mass spectrometry imaging (MSI) in microprobe mode involves desorption of the sample from a surface by a beam (laser or ions) at discrete locations over the surface of a sample. At each raster point, a mass spectrum is generated and the acquired spectra, along with the spatial location from which they were collected, can be used to simultaneously map numerous analytes within the sample. This label-free manner of imaging coupled to the sensitivity and specificity of mass spectrometry have helped MSI become one of the most rapidly evolving fields in mass spectrometry1,2.
Matrix-assisted laser desorption/ionization (MALDI) is the most common ionization method used for MSI analyses. However, the need for an organic matrix and the vacuum requirements of MALDI pose significant limitations on reproducibility, sample throughput, and the types of samples that can be analyzed using the method. A number of atmospheric pressure (AP) ionization methods have been developed in recent years to circumvent these restrictions3. These ambient ionization methods allow for analysis of biological samples in an environment that is much closer to their natural state and simplify sample preparation steps prior to analysis. Matrix-assisted laser desorption electrospray ionization (MALDESI) is an example of such an ionization method4,5.
In IR-MALDESI experiments, a thin layer of ice is deposited on the tissue surface as the energy-absorbing matrix. A mid-IR laser pulse is absorbed by the ice matrix, and facilitates desorption of neutral materials from the surface by resonantly exciting the O-H stretching mode of water. The desorbed neutrals partition into the charged droplets of an orthogonal electrospray and are post-ionized in an ESI-like fashion4-6. The addition of exogenous ice matrix is preferred over relying solely on the endogenous water in tissue since it helps account for variations in water content in different tissue compartments, and has been shown to enhance desorption6 and improve ion abundance by ~15-fold7,8 in tissue imaging experiments.
In this work, we utilize IR-MALDESI MSI to elicit the distribution of metabolites across different organs in a neonatal mouse whole body. An overview of adjustable parameters of the IR-MALDESI source is given, and the necessary steps for successful imaging of tissue sections are demonstrated.

<table border="0" cellpadding="0" cellspacing="0" width="963">
	<tbody>
		<tr height="69">
			<td height="69" style="height:69px;width:283px;">IR-MALDESI Source</td>
			<td style="width:188px;">Custom-made</td>
			<td style="width:171px;">N/A</td>
			<td style="width:323px;">Please refer to references 4 and 12 for an in-depth discussion of IR-MALDESI source development.</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:283px;">Q Exactive Plus&#160;</td>
			<td style="width:188px;">Thermo Scientific</td>
			<td style="width:171px;"></td>
			<td style="width:323px;">Q Exactive Plus Hybrid Quadrupole-Orbitrap Mass Spectrometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Water, HPLC Grade</td>
			<td style="width:188px;">Burdick &#38; Jackson&#160;</td>
			<td style="width:171px;">AH365-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Methanol, HPLC Grade</td>
			<td style="width:188px;">Burdick &#38; Jackson&#160;</td>
			<td style="width:171px;">AH230-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Formic Acid</td>
			<td style="width:188px;">Sigma Aldrich&#160;</td>
			<td style="width:171px;">56302</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Tunable mid-IR Laser</td>
			<td style="width:188px;">Opotek Inc.</td>
			<td style="width:171px;">IR Opolette</td>
			<td>Tunable 2700-3100 nm IR OPO laser</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Nitrogen Gas</td>
			<td style="width:188px;">Arc3 Gases</td>
			<td style="width:171px;">AG S-NI300-5.0</td>
			<td>Grade 5.0 high purity nitrogen gas cylinder (300)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Cryostat</td>
			<td style="width:188px;">Leica Biosystems</td>
			<td style="width:171px;">CM 1950</td>
			<td>Cryomicrotome</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">High Profile Microtome Blades</td>
			<td style="width:188px;">Leica Biosystems</td>
			<td>3802123</td>
			<td>Leica DB80HS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Mounting Medium (OCT)</td>
			<td style="width:188px;">Leica Biosystems</td>
			<td style="width:171px;">3801480</td>
			<td>Surgipath FSC 22 mounting medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Cryostat Specimen Disc</td>
			<td style="width:188px;">Leica Biosystems</td>
			<td style="width:171px;">14047740045</td>
			<td>40 mm diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Glass Microscope Slides</td>
			<td style="width:188px;">VWR</td>
			<td style="width:171px;">48312-003</td>
			<td>Frosted, selected, pre-cleaned</td>
		</tr>
	</tbody>
</table>

whole-body mass spectrometry imaging by infrared matrix-assisted laser desorption electrospray ionization (ir-maldesi)

Ambient ionization sources for mass spectrometry (MS) have been the subject of much interest in the past decade. Matrix-assisted laser desorption electrospray ionization (MALDESI) is an example of such methods, where features of matrix-assisted laser desorption/ionization (MALDI) (e.g., pulsed nature of desorption) and electrospray ionization (ESI) (e.g., soft-ionization) are combined. One of the major advantages of MALDESI is its inherent versatility. In MALDESI experiments, an ultraviolet (UV) or infrared (IR) laser can be used to resonantly excite an endogenous or exogenous matrix. The choice of matrix is not analyte dependent, and depends solely on the laser wavelength used for excitation. In IR-MALDESI experiments, a thin layer of ice is deposited on the sample surface as an energy-absorbing matrix. The IR-MALDESI source geometry has been optimized using statistical design of experiments (DOE) for analysis of liquid samples as well as biological tissue specimens. Furthermore, a robust IR-MALDESI imaging source has been developed, where a tunable mid-IR laser is synchronized with a computer controlled XY translational stage and a high resolving power mass spectrometer. A custom graphical user interface (GUI) allows user selection of the repetition rate of the laser, number of shots per voxel, step-size of the sample stage, and the delay between the desorption and scan events for the source. IR-MALDESI has been used in variety of applications such as forensic analysis of fibers and dyes and MSI of biological tissue sections. Distribution of different analytes ranging from endogenous metabolites to exogenous xenobiotics within tissue sections can be measured and quantified using this technique. The protocol presented in this manuscript describes major steps necessary for IR-MALDESI MSI of whole-body tissue sections.

The images presented in Figure 4 show the spatial distribution of metabolites in different organs in the whole-body tissue section. Unique m/z values to specific regions of the body were found using MSiReader PeakFinder, followed by batch processing for image generation. The image overlay tool (Figure 3-4) was used to align the optical image taken before ice matrix deposition with the resulting ion maps. Cholesterol is observed across all tissue ...

Watch this Scientific Journal Video about Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization IR-MALDESI at JoVE.com

Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization IR-MALDESI

A mass spectrometry imaging (MSI) source operated at atmospheric pressure was developed by coupling mid-infrared laser desorption and electrospray post-ionization. Exogenous ice matrix was used as the energy-absorbing matrix to facilitate resonant desorption of tissue-related material. This manuscript provides a step-by-step protocol for performing IR-MALDESI MSI of whole-body neonatal mouse.

Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization (IR-MALDESI)

Ambient ionization sources for mass spectrometry (MS) have been the subject of much interest in the past decade. Matrix-assisted laser desorption ...

The overall goal of this protocol is to describe the major steps necessary for IR-MALDESI mass spectrometry imaging of whole body tissue sections. This method can effectively answer key questions in the field of mass spectrometry imaging, such as discerning the spatial distribution of endogenous and exogenous analytes in multiple organs simultaneously. The main advantage of this technique is that it operates at atmospheric pressure and the exogenous ice matrix helps account for variations in water content in different organs.Although this protocol provides insight into a targeted whole body imaging. IR-MALDESI mass spectrometry imaging can also be applied to targeted and quantitative drug distribution analysis. The following protocol describes how to perform IR-MALDESI MSI experiments.In depth details about the optimized geometry of the IR-MALDESI source and it's synchronization with the laser, stage, and mass spectrometer can be found elsewhere. To prepare the specimen, wear protective gloves, and apply a layer of OCT mounting medium onto a 40 millimeter cryostat specimen disc. Then, gently place a frozen whole mouse specimen onto the OCT coated holder and orient it for sectioning.OCT is used solely to adhere the tissue to the specimen disc. Do not completely embed the tissue in the OCT. Place the specimen disc on the Peltier sample holder in the cryostat.Turn on the Peltier and wait 10 minutes for the sample to equilibrate. Then, place the specimen disc inside the disc holder, orient it to slice to the desired plane. Section the specimen at 20 degrees Celsius at, for instance, 25 micorons for whole body analysis.Make use of the anti-roll glass plate and use the cryostat vacuum and brushes to remove unwanted tissue debris. After the tissue is sectioned, cover the blade with a blade guard. Now, orient the frozen section on the specimen plate and thaw mount them onto pre-cleaned glass microscope slides.Bring the slides as close as possible to the sections without touching. Keep the mounted slides inside the cryostat until the IR-MALDESI calibration is complete to maintain the tissues cryopreservation. To begin, turn on the mid-IR laser and launch the laser control application.Then, choose the desired wavelength. IR-MALDESI experiments are typically performed at 2940 nanometers. Next, turn on the stage controller and launch the control program.Using the program, calibrate the stage position using the home button. Next, fill a one milliliter syringe with electrospray solvent and flush the silica capillary. Then, align the ESI emitter with the MS inlet using the source parameters, such as ESI-spot distance, spot-inlet distance, sample height, and solvent flow rate.All of which have been optimized using statistical DOE for analysis in tissue sections. Now start the electrospray and evaluate it's stability for at least 10 minutes. Monitor the total ion current, which at this point, consists solely of ambient compounds.Variation under 10%over a 10-15 minute period indicates the system is stable. After turning off the electrospray and ensuring it is off, transfer a specimen slide from the cryostat to the IR-MALDESI sample plate. Then, stand back from the ESI emitter and restart the electrospray.Close the access door of the IR-MALDESI source and purge the enclosure with dry nitrogen to prevent condensation of water on the tissue. Once the relative humidity inside the enclosure is less than 3%switch on the Peltier plate's DC power supply to about 12 volts to cool the sample plate to 9 degrees Celsius. After about 5-10 minutes, stop the nitrogen purge and open the source access door to expose the tissue to the ambient humidity.A thin layer of ice will form on the stage and mounted tissue section. If the air is dry, place a beaker of warm water inside the enclosure. Now, close the door and restart the nitrogen flow to reduce the humidity to a around 10%Adjust the nitrogen flow as needed to maintain a stable layer of ice on the sample during the experiment.Using the Rastier GUI, move the stage to the analysis position by clicking the LASER Position button. Use the adjustment diode of the laser to ensure an off tissue region will be abladed. Then, fire a test shot to calibrate the laser's offset with respect to the camera view.Click the Laser Test Fire button to update the laser position. Now, return to camera position and update the laser position in Rastier by placing the laser alignment reticle on the laser ablation spot. Then, click the ROI button and select the region of interest.Include some off tissue area to serve as control. Use this off tissue section for peak picking. The spot size of the laser on tissue is about 150 microns.A smaller diameter is possible with over sampling. Now, input the appropriate experimental parameters and name the mass spectrometer imaging file in the project name box. Then, select the appropriate laser frequency from the drop down box.Typically, use two laser pulses per voxel at 20 Hertz to completely desorb the material. Also, set the delay time to 10 milliseconds so the ions have time to reach the mass analyzer. The repetition rate should be set to the laser's maximum.Now, choose mass spectrometer parameters, such as ionization mode, electrospray voltage, solvent flow rate, capillary temperature, and the injection time in the mass spectrometer software. Once all parameters have been set, place the mass spectrometer in handshake mode for synchronization and start the mass spectrometer acquisition. Using the Rastier imaging software, verify that all steps in the checklist have been completed and then load the program.Once the RUN button becomes available, start the MSI signal acquisition. Upon completion of the imaging experiment, stop mass spectrometer acquisition and place the instrument in standby mode. Turn off the nitrogen purge to the enclosure.Turn off the stage controller. Turn off the power supply to the Peltier plate and turn off the laser. Then, open the access door.Remove the eletrospray emitter tip from the inside of the source enclosure and wearing protective gloves, remove the very hot extended metal mass spectrometer inlet. Wearing gloves and safety goggles, clean the inlet using an ultrasonic bath. First, use 15%nitric acid for 10 minutes.Then, use HPLC grade water for 10 minutes and finally, use HPLC grade methanol for 10 minutes. Dry the metal capillary inlet under a stream of nitrogen and reinsert it into the mass spectrometer for future analysis. Spatial distribution of metabolites in different organs of whole body tissue sections were analyzed using the described method.Cholesterol was observed in all tissues because of it's structural role in cell membranes. Other lipid metabolites exhibited distinct distributions within specific organs or tissue compartments. For example, some lipids, such as punitively assigned desmosterol were observed to have a specific localization in the brain.Whereas other lipids exhibited a higher distribution in the spinal cord. And other lipids were uniforming distributed across all organs except for the brain. IR-MALDESI mass spectrometry imaging is a disruptive technology that allows for the relative and absolute quantification of analytes in tissues with minimal sample preparation at atmospheric pressure.Once mastered, all of the sample preparation steps can be done in approximately 30 minutes. A key step in sample preparation is the formation of the ice matrix, which facilitates efficient desorption of tissue related material. The ice matrix also helps account for variations in water content in different organs.In tandem with this protocol, supplementary analysis, such as histological and immunohistochemical staining of the serial section can be performed.

whole-body-mass-spectrometry-imaging-infrared-matrix-assisted-laser

Research

JoVE Journal

Bioengineering

Characterizing Bacterial Volatiles using Secondary Electrospray Ionization Mass Spectrometry (SESI-MS)

Secondary electrospray ionization mass spectrometry (SESI-MS) is a method developed for the rapid detection of volatile compounds, without the need for sample pretreatment. The method was first described by Fenn and colleagues1 and has been applied to the detection of illicit drugs2 and explosives3-4, the characterization of skin volatiles5, and the analysis of breath6-7.
SESI ionization occurs by proton transfer reactions between the electrospray solution and the volatile analyte, and is therefore suitable for the analysis of hetero-organic molecules, just as in traditional electrospray ionization (ESI). However, unlike standard ESI, the proton transfer process of SESI occurs in the vapor phase rather than in solution (Fig. 1), and therefore SESI is best suited for detecting organic volatiles and aerosols.
We are expanding the use of SESI-MS to the detection of bacterial volatiles as a method for bacterial identification and characterization8. We have demonstrated that SESI-MS volatile fingerprinting, combined with a statistical analysis method, can be used to differentiate bacterial genera, species, and mixed cultures in a variety of growth media.8 Here we provide the steps for obtaining bacterial volatile fingerprints using SESI-MS, including the instrumental parameters that should be optimized to ensure robust bacterial identification and characterization.

Characterizing Bacterial Volatiles using Secondary Electrospray Ionization Mass Spectrometry SESI-MS

Secondary electrospray ionization mass spectrometry (SESI-MS) enables the detection of volatile organic compounds (VOCs) without the need for any sample pretreatment. This protocol provides instructions for the rapid (within minutes) characterization of bacterial VOCs using SESI-MS.

Secondary electrospray ionization mass spectrometry (SESI-MS) is a method developed for the rapid detection of volatile compounds, without the need for ...

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry

Mass spectrometry imaging (MSI) provides untargeted molecular information with the highest specificity and spatial resolution for investigating biological tissues at the hundreds to tens of microns scale. When performed under ambient conditions, sample pre-treatment becomes unnecessary, thus simplifying the protocol while maintaining the high quality of information obtained. Desorption electrospray ionization (DESI) is a spray-based ambient MSI technique that allows for the direct sampling of surfaces in the open air, even in vivo. When used with a software-controlled sample stage, the sample is rastered underneath the DESI ionization probe, and through the time domain, m/z information is correlated with the chemical species' spatial distribution. The fidelity of the DESI-MSI output depends on the source orientation and positioning with respect to the sample surface and mass spectrometer inlet. Herein, we review how to prepare tissue sections for DESI imaging and additional experimental conditions that directly affect image quality. Specifically, we describe the protocol for the imaging of rat brain tissue sections by DESI-MSI.

Desorption electrospray ionization mass spectrometry (DESI-MS) is an ambient method by which samples, including biological tissues, can be imaged with minimal sample preparation. By rastering the sample below the ionization probe, this spray-based technique provides sufficient spatial resolution to discern molecular features of interest within tissue sections.

Mass spectrometry imaging (MSI) provides untargeted molecular information with the highest specificity and spatial resolution for investigating biological ...

Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with some of the spatial and molecular complexity of tissue. For example, many cell lines form 3D structures given appropriate in vitro conditions. Colon cancer cell lines form 3D cell culture spheroids, in vitro mimics of avascular tumor nodules. While immunohistochemistry and other classical imaging methods are popular for monitoring the distribution of specific analytes, mass spectrometric imaging examines the distribution of classes of molecules in an unbiased fashion. While MALDI mass spectrometric imaging was originally developed to interrogate samples obtained from humans or animal models, this report describes the analysis of in vitro three dimensional cell cultures, including improvements in sample preparation strategies. Herein is described methods for growth, harvesting, sectioning, washing, and analysis of 3D cell cultures via matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging. Using colon carcinoma 3D cell cultures as a model system, this protocol demonstrates the ability to monitor analytes in an unbiased fashion across the 3D cell culture system with MALDI-MSI.

Immortalized cancer cell lines can be grown as 3D cell cultures, a valuable model for biological research. This protocol describes mass spectrometry imaging of 3D cell cultures, including improvements in the sample preparation platform. The goal of this protocol is to instruct users to prepare 3D cell cultures for mass spectrometry imaging analysis.

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with ...

Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem cell (iPS) emerged as a new method in regenerative medicine. Although this tissue regeneration is promising, contamination with unwanted cells during tissue cultures is a major concern. Moreover, there is a safety concern regarding tumorigenicity after transplantation. Therefore, there is an urgent need for eliminating specific cells without damaging other cells that need to be protected, especially in established tissue. Here, we present a method for specific cell elimination from a mixed 3D cell culture in vitro with near infrared photoimmunotherapy (NIR-PIT) without damaging non-targeted cells. This technique enables the elimination of specific cells from mixed cell cultures or tissues.

Eliminating specific cells without damaging other cells is extremely difficult, especially in established tissue, yet there is an urgent need for a cell elimination method in the tissue engineering field. Here, we present a method for specific cell elimination from a mixed 3D cell culture using near infrared photoimmunotherapy (NIR-PIT).

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem ...

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and engineered variants of the green fluorescent protein from Aequorea victoria (avGFP) as well as homologs from other species already cover large parts of the optical spectrum, a spectral gap remains in the near-infrared region, for which avGFP-based fluorophores are not available. Red-shifted fluorescent protein (FP) variants would substantially expand the toolkit for spectral unmixing of multiple molecular species, but the naturally occurring red-shifted FPs derived from corals or sea anemones have lower fluorescence quantum yield and inferior photo-stability compared to the avGFP variants. Further manipulation and possible expansion of the chromophore&#39;s conjugated system towards the far-red spectral region is also limited by the repertoire of 20 canonical amino acids prescribed by the genetic code. To overcome these limitations, synthetic biology can achieve further spectral red-shifting via insertion of non-canonical amino acids into the chromophore triad. We describe the application of SPI to engineer avGFP variants with novel spectral properties. Protein expression is performed in a tryptophan-auxotrophic E. coli strain and by supplementing growth media with suitable indole precursors. Inside the cells, these precursors are converted to the corresponding tryptophan analogs and incorporated into proteins by the ribosomal machinery in response to UGG codons. The replacement of Trp-66 in the enhanced &#34;cyan&#34; variant of avGFP (ECFP) by an electron-donating 4-aminotryptophan results in GdFP featuring a 108 nm Stokes shift and a strongly red-shifted emission maximum (574 nm), while being thermodynamically more stable than its predecessor ECFP. Residue-specific incorporation of the non-canonical amino acid is analyzed by mass spectrometry. The spectroscopic properties of GdFP are characterized by time-resolved fluorescence spectroscopy as one of the valuable applications of genetically encoded FPs in life sciences.

Engineering &#039;Golden&#039; Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Synthetic biology enables the engineering of proteins with unprecedented properties using the co-translational insertion of non-canonical amino acids. Here, we presented how a spectrally red-shifted variant of a GFP-type fluorophore with novel fluorescence spectroscopic properties, termed &#34;gold&#34; fluorescent protein (GdFP), is produced in E. coli via selective pressure incorporation (SPI).

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and ...

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy (oSLO) and Optical Coherence Tomography (OCT)

While fluorescence imaging is widely used in ophthalmology, a large field of view (FOV) three-dimensional (3D) fluorescence retinal image is still a big challenge with the state-of-the-art retinal imaging modalities because they would require z-stacking to compile a volumetric dataset. Newer optical coherence tomography (OCT) and OCT angiography (OCTA) systems overcome these restrictions to provide three-dimensional (3D) anatomical and vascular images, but the dye-free nature of OCT cannot visualize leakage indicative of vascular dysfunction. This protocol describes a novel oblique scanning laser ophthalmoscopy (oSLO) technique that provides 3D volumetric fluorescence retinal imaging. The setup of the imaging system generates the oblique scanning by a dove tail slider and aligns the final imaging system at an angle to detect fluorescent cross-sectional images. The system uses the laser scanning method, and therefore, allows an easy incorporation of OCT as a complementary volumetric structural imaging modality. In vivo imaging on rat retina is demonstrated here. Fluorescein solution is intravenously injected to produce volumetric fluorescein angiography (vFA).

Multimodal Volumetric Retinal Imaging by Oblique Scanning Laser Ophthalmoscopy oSLO and Optical Coherence Tomography OCT

Here, we present a protocol to get a large field of view (FOV) three-dimensional (3D) fluorescence and OCT retinal image by using a novel imaging multimodal platform. We will introduce the system setup, the method of alignment, and the operational protocols. In vivo imaging will be demonstrated, and representative results will be provided.

While fluorescence imaging is widely used in ophthalmology, a large field of view (FOV) three-dimensional (3D) fluorescence retinal image is still a big ...

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...