Ye He and Rinat Abzalimov are supported by the Professional Staff Congress-City University of New York (PSC-CUNY) Research Award Program. Yuki Chen and Kelly Veerasammy are supported by the Alfred P. Sloan Foundation CUNY Summer Undergraduate Research Program.

The protocol follows the guidelines of City University of New York (CUNY) Advanced Science research Center (ASRC)&#39;s institutional animal care and use committee (IACUC).
1. Harvest the tissue
<ol>
	<li>Prepare the aluminum boat. Prepare a rectangle aluminum 10cm x 20 cm, fold in the middle to make 10 cm double layer square. Label the sample information on one side, and fold the other side to form a boat with bottom surface about 4 cm x 4 cm.</li>
	<li>Precool the boat on the liquid nitrogen (LN2) in a Styrofoam box. 
	&#8203;NOTE: If the boat is too small, the sample might fall out of the boat and become over frozen by directly contacting LN2, which may result in fragmentation during cryosectioning.</li>
	<li>Euthanize the animal by cervical dislocation following institutional IACUC guidelines, immediately dissect out the tissue of interest.
	<ol>
		<li>Keep the interval between animal anesthesia and snap freezing as short as possible, to minimize the alteration of metabolites during tissue harvesting, especially the labial metabolites in the brain due to postmortem ischemia. 
		NOTE: Perfusion the animal with phosphate-buffered saline (PBS) will increase the duration of ischemia, further altering the concentrations of labile metabolites and exaggerating the artifactual results. Therefore, perfusion or washing the tissue with PBS is not recommended, unless the contamination of blood or body fluid is of more concerned than metabolites deterioration or washout for individual project.</li>
	</ol>
	</li>
	<li>Immediately place the tissue into the aluminum boat floating on liquid nitrogen, close the lid of Styrofoam box, and freeze for 2-10 min depending on the size of the tissue: 2 min, 5 min, 7 min for postnatal day 1 (P1), P21, P60 mouse brain, respectively, and 10 min for P60 rat brain. 
	NOTE: Do not freeze the tissue for prolonged time (e.g., 5 min for P1 mouse brain) as the over-frozen might lead to tissue fragmentation during cryosectioning.</li>
	<li>Remove the boat by forceps, fold the foil to wrap the tissue, transport on dry ice and store at -80 &#176;C for later use. If followed by immediate sectioning, carry the samples on dry ice to the cryostat. 
	&#8203;NOTE: To better preserve the metabolites, it is preferred to store the sample in intact tissue and section it right before proceeding to MALDI imaging. The tissue can be stored in -80 &#176;C for up to 24 months.</li>
</ol>
2. Cryosection the tissue
NOTE: Wear gloves at all times when handling the Indium tinoxide (ITO) slides. Avoid direct breathing onto the slide or wear masks (optional) to prevent the contamination of human saliva onto the tissue section.
<ol>
	<li>Before sectioning the tissue, gather the desired number of MALDI compatible ITO coated glass slides.</li>
	<li>Test the conductivity of the slide using a voltmeter set to resistance. Label the side where a resistance measurement is read: this will be the side that the tissue sections adhere to. Always place the slide on a clean paper towel to avoid contamination.</li>
	<li>Pre-cool the slides in a cryostat set to -20 &#176;C.</li>
	<li>If tissue samples were removed from the -80 &#176;C, let the tissue equilibrate in the cryostat chamber for about 45-60 min depending on the size of the tissue. If samples are removing from dry ice, equilibrate it for about 30 min.</li>
	<li>Thoroughly clean the cryostat with 70% ethanol. Pre-cool all the necessary tools including thin-tipped artist brush and forceps in the cryostat chamber.</li>
	<li>Set the temperature of the cryostat chamber and specimen head according to the type of the tissue: -14 &#176;C for liver, -20 &#176;C for muscle and -25 &#176;C for skin9.</li>
	<li>Mount the tissue to the chuck using cryo tissue embedding compound OCT, avoiding OCT from the region of interest.</li>
	<li>Place a clean blade in the stage and lock. Adjust the position of the stage and the angle of the specimen to achieve the desired cutting angle. 
	NOTE: If different types/genotypes of tissue are to be cut, be sure to either reposition the sample so that a clean part of the blade is used , or change to a new blade before cutting the next sample to prevent cross-contamination.</li>
	<li>Continue cutting until the region of interest (e.g., corpus callosum in the brain) is found. Be sure to keep the stage clean by brushing off extra pieces with an artist brush that has been equilibrated in the cryostat.</li>
	<li>Once the desired region is revealed, cut smaller sections of 10-12 &#181;m thickness. If the section tends to flake or fall apart easily, raise the temperature of the cryostat, staying in the -22 &#176;C to -11 &#176;C range. We have found that the optimal cutting temperature for brain tissue is -15 &#176;C to - 18 &#176;C.</li>
	<li>Once a good section has been cut, adhere it to the ITO slide (operate in the cryostat chamber).</li>
	<li>Transfer one section of the tissue onto ITO slides using the tip of artist brush.</li>
	<li>Finger-warm the section by placing a finger under the slide to warm up the section to ensure secure mounting. The tissue section will first turn to transparent in 5-10 s and then turn opaque in about 30-60 s.</li>
	<li>Carefully set the slide aside in the cryostat.</li>
	<li>Repeat steps for other tissue samples, ensuring that each section&#160;of the tissue is placed evenly on the slide and is as aligned as possible.</li>
	<li>Since the MALDI target can hold up to two slides, place the sections from multiple samples of the same cohort onto a single slide or onto two slides, to ensure they can be analyzed at the same time.</li>
	<li>When finished, place ITO slides in a vacuum box and transfer to a desiccator with desiccant. Vacuum-dry the slide for 45-60 min. 
	NOTE: If a vacuum desiccator is not available in the lab, keep the slides under -20 &#176;C all the time to avoid metabolites deterioration.</li>
	<li>Slide storage and shipping: unless the sample is prepared by the MALDI imaging core facilities to proceed directly to MALDI imaging, store the slides at -80 &#176;C or ship to core facilities or other MALDI research laboratories on dry ice.</li>
	<li>To best preserve the samples, place the slides into the slide transporter, fill it with nitrogen (optional), seal with parafilm, place into a zip bag, which is then placed into another zip bag containing desiccant. Label the outer zip bag.</li>
	<li>Proceed to storage at -80 &#176;C (up to 6 months) or shipping with adequate dry ice.</li>
</ol>
3. Matrix Preparation
<ol>
	<li>Prepare the matrix. 
	&#8203;NOTE: All the reagents must be HPLC grade.
	<ol>
		<li>Prepare NEDC at a concentration of 10 mg/mL. Prepare 10 mL of matrix dissolved in a solvent of 70% methanol (100 mg of NEDC, 7 mL of methanol, 3 mL of H2O).</li>
		<li>Additionally prepare an extra 10 mL of 70% methanol:water solution to flush the sprayer system before filling in the matrix.</li>
	</ol>
	</li>
	<li>Once the slides are dehydrated in step 2.17, place &#34;X&#34; marks on the blank space of the glass slide outside of the tissue sections using a bold point silver marker, and then place a second &#34;x&#34; with a fine point black marker on top of the silver &#34;X&#34;. The black &#34;X&#34; with a sharp contrast out of the silver background (the bold silver &#34;X&#34;) will later serve as fiduciary marker for the later mass spectra acquisition in MALDI instrument.</li>
	<li>Load the slide into the MALDI slide metal target. Use plastic cover and draw/outline where the samples are onto the plastic cover. Set aside.</li>
	<li>Scan the image of the slide together with the MALDI target using flatbed scanner. The screws on the surface of the target will serve as the spacer to prevent the damage or contamination of the tissue section by the scanner. Preview and scan the selected slide area in 16-bit grayscale and 2400 dpi. Save the image for later use in MALDI MSI.</li>
</ol>
4. Matrix deposition
NOTE: There are multiple methods to apply an even layer of matrix in fine crystal size on to the MALDI slide, including sublimation, droplet inkjet printing, automatic matrix sprayer and manual spray using artist airbush9. We will use automatic matrix sprayer as an example in this protocol for its high reproducibility.
<ol>
	<li>Start up: Turn on the matrix sprayer unit, being sure that the valve is positioned at LOAD and launch the sprayer software. Check that the exhaust fan is operating well. Do not start the solvent pump if the active venting is not functioning properly.</li>
	<li>Confirm on the Comms tab that everything is communicating, and then start the solvent pump at .1 mL/min, with a backpressure of ~500 psi (or 3.4 MPa).</li>
	<li>Start compressed air flow to the matrix sprayer by setting the nitrogen tank to 30 psi. Then, adjust the pressure regulator on the front of the sprayer to 10 psi, and set sprayer nozzle temperature as desired. 
	NOTE: If the air pressure in prayer is lower than 5 psi, the sprayer nozzle will not be able to heat up for protection.</li>
	<li>With the valve still in the LOAD position, use a syringe to flush the loop with 7 mL of 70% methanol, and then fill the loop with 6 mL of matrix.</li>
	<li>Place the tissue slides into the holders in the sprayer, taping down both ends to prevent movement, and to preserve a matrix-free edge to avoid the contamination of the metal clamp on MALDI target by the matrix. 
	NOTE: Testing matrix spray on a blank microscope slide first before proceeding to precious sample slides is highly recommended.</li>
	<li>Check that the flow rate and temperature are stable to begin spraying.</li>
	<li>Select desired method pre-tested using blank glass slide.</li>
	<li>Press Start. This will set the nozzle temperature and adjust the pump flow rate to match the selected method. Switch the valve&#160;from Load to Spray position then confirm by clicking Continue.</li>
	<li>Allow the system to run, which will take 5-20 min per slide depending on the method. Switch the valve from Spray to Load position then confirm by clicking Continue when finished.</li>
	<li>Remove the slide(s) from the sprayer, examine the pattern of matrix coating under microscope to ensure an even layer of fine matrix crystal.</li>
	<li>After matrix deposition, place the slide(s) into the metal MALDI holder for immediate use. If there is only one specimen slide, add another blank slide to fill the two spaces of the MALDI holder.</li>
	<li>Clean up the system immediately after the usage following the manufacturer&#39;s instruction, to prevent the clogging of sprayer nozzle.</li>
	<li>After the sample slide is coated with matrix, either proceed immediately to MALDI imaging instrument, or ship it with dry ice using the same double zip bag preparation described in step 2.19. 
	NOTE: Under emergency circumstance such as the MALDI instrument is not available for immediate usage, store the coated slide in vacuum condition or at -20 &#176;C for up to 24 hrs, although the deterioration of some metabolites might happen during the storage, which has not been thoroughly studied.</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolomic+Assays+of+Postmortem+Brain+Extracts:+Pitfalls+in+Extrapolation+of+Concentrations+of+Glucose+and+Amino+Acids+to+Metabolic+Dysregulation+In+Vivo+in+Neurological+Diseases.">Metabolomic Assays of Postmortem Brain Extracts: Pitfalls in Extrapolation of Concentrations of Glucose and Amino Acids to Metabolic Dysregulation In Vivo in Neurological Diseases.</a> Neurochemistry Research. 44, 2239-2260 (2019).</li><li>Wasek, B., Arning, E., Bottiglieri, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+microwave+irradiation+for+quantitative+analysis+of+neurotransmitters+in+the+mouse+brain.">The use of microwave irradiation for quantitative analysis of neurotransmitters in the mouse brain.</a> Journal of Neuroscience Methods. 307, 188-193 (2018).</li><li>Andres, D. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Absolute+Quantification+of+Rifampicin+by+MALDI+Imaging+Mass+Spectrometry+Using+Multiple+TOF/TOF+Events+in+a+Single+Laser+Shot.">Absolute Quantification of Rifampicin by MALDI Imaging Mass Spectrometry Using Multiple TOF/TOF Events in a Single Laser Shot.</a> Journal of the American Society for Mass Spectrometry. 28 (1), 136-144 (2017).</li><li>Quanico, J., Franck, J., Wisztorski, M., Salzet, M., Fournier, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+MALDI+Mass+Spectrometry+Imaging+and+Parafilm-Assisted+Microdissection-Based+LC-MS/MS+Workflows+in+the+Study+of+the+Brain.">Combined MALDI Mass Spectrometry Imaging and Parafilm-Assisted Microdissection-Based LC-MS/MS Workflows in the Study of the Brain.</a> Methods in Molecular Biology. 1598, 269-283 (2017).</li><li>Trim, P. 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The authors declare no competing financial interests.

MALDI-Imaging (MALDI-MSI) is a label-free imaging technique that allows researchers to investigate the distribution of various biomolecules and their modifications in tissue, the molecular basis of pathology. Combined use of MALDI-MSI with traditional LC-MS approaches for tissue analysis provides the same molecular depth as traditional Omics workflows but which also retains the spatial relationship of those signals within the cellular network. The sample preparation is the most critical step in MALDI MSI and accounts for the variation in the final read outs of metabolomics studies carried out in different labs4. Here we provide a comprehensive yet practical protocol to standardize the sample preparation for metabolomic profiling using MALDI MSI, in the hope that it&#160;benefits a broad research community to implement MALDI MSI in their current and future research from basic biology to translational studies.
One must always bear in mind all precautions to minimize changes in the molecular profiles (both abundances and spatial distribution) during sample preparations and avoid contamination. Firstly, minimize the time between animal euthanasia and tissue harvesting, such as frozen in situ or heated by microwave fixation to inactivate enzymes in the brain to reduce the liability to postmortem ischemia4,5,6. Secondly, the snap freezing condition of the sample is critical. Inadequate freezing will cause the degradation and loss of the metabolites, while over freezing will lead to tissue fragmentation during cryosectioning. The freezing time should always be tested first according to previous reported studies, and the study of developmental mouse brain presented in this paper provides the reference points for rodent brain tissue. Thirdly, cutting of biological tissues and transferring their sections to the ITO slides will require adequate practice. It is important to note that while using the brush to pick up the section from the stage, the brush should be used delicately. Allow the bristles of brush to only come in contact with the edges of the tissue section to decrease the risk of contamination and section fragmentation. Flatten the section on the slide as much as possible, this will prevent the curling of the section during finger-warming. Fourthly, while mounting onto the ITO slide, make sure the entire section is well attached to the ITO slide as different regions of the tissue might require different time of finger-warming. For example, brain tumor tissue requires longer warming time than normal brain tissue. A poor mounting might lead to detachment and fragmentation of the tissue during MALDI-MS scanning. Bear in mind that the finger warming might enable enzyme action and metabolism causing artifactual changes of metabolites. Fifthly, a fine and uniform deposition of MALDI matrix plays an important role in achieving accurate spatial information and strong MALDI-MS signal. It is recommended to test the matrix spraying on a blank slide, and observe the crystal pattern under a microscope to verify proper coverage, before proceeding to the coating of precious specimen slide. And lastly, since an individual researcher executes the tissue harvesting and slide preparation at different speeds, it would be ideal to have one person to handle the sample preparation for the specimen in the same cohort, to minimize the variation.
The protocol provided above details standard procedures, which can be tailored towards the needs of particular experiments. For example, a cryo-sectioning gel OCT, which is normally used in cryo-sectioning of the sample, can be further utilized as a mounting glue to the tissue chuck (as in the study described above). Prior studies have shown that the polymer component in OCT causes strong ion suppression14. However, embedding the sample may be unavoidable in the cases where the tissue is too fragile to be cut without additional support from a polymer gel. In order to combat signal suppression in these cases, the tissues may need to be washed with serial washing in 70% ethanol and 95% ethanol to remove residual OCT for the detection of proteins or lipids, while the washing is not recommended for the detection of small molecular metabolites9.
MALDI MSI has grown increasingly relevant in both the research laboratory and clinical practice setting. For instance, MALDI MSI has recently proved useful in studies of proteomics in order to characterize the phenotypic functional status of an organism15, and in acting as an agent for microbial identification and diagnosis of subsequent disease16. While MALDI MSI supports a wide range of applications, there are some limitations associated with relying solely on this technique, especially when it comes to differentiating between similar species or metabolites, and the identification of specific targets. Another challenge is the quantification of the metabolites concentration according to MALDI MSI signals. It is often assumed that ion abundances in MALDI MSI spectra and the spatial distribution (or relative abundances) of corresponding molecular species across dissected tissues are well correlated. However, one should always bear in mind that the relationship between ion intensity and the amount of corresponding molecular species is complicated by numerous factors including, but not limited to, effects of ion suppression, changes in tissue structure and&#160;ion-molecule reactions17. Techniques that make use of internal standards can be implemented for absolute quantification (&#181;mol/g tissue) in MALDI-MSI18. These two challenges are typically addressed with the combined workflow of MALDI MSI with liquid chromatography tandem MS(LC-MS/MS) techniques, whereby MALDI-MS allows for the mapping of the region of interest, which is later subjected to microextraction and LC-MS/MS to provide more information for identifying the metabolite19.
MS-based imaging methods have been developed in recent years as an alternative modality to previous techniques for imaging small molecule metabolites. With the advancements and growing popularity of MALDI MSI, it is expected that MALDI imaging will become a new standard tool for visualizing small molecules. Imaging of lipid and endogenous small molecules (e.g. neurotransmitters and metabolites) in the biological context, as well as imaging of xenobiotics for new pharmaceutical agents development are of particular interest. These three areas are expected to have rapid advancements with application of MALDI MSI in the near future20.

Metabolites, the intermediates or end products of metabolism, including nucleotides, amino or organic acids, lipids, are key components to biological functions and processes. Metabolomics, the study of metabolites, allows for the exploration of their biochemical interactions and the understanding of their roles in the context of basic, translational, and clinical research. Metabolites are strongly associated with the phenotypes of organisms and provide information on biochemical activities that occur during cellular metabolism1. Therefore, in addition to genomics and proteomics, metabolomics has emerged as an important tool in understanding both physiological and pathological conditions. For instance, metabolomics is used in elucidating the mechanisms behind existing drugs as well as their tolerance. In drug development, xenobiotic metabolism is useful in assessing the activity or toxicity of metabolites across species, which later translates to supporting personalized medicine2. Despite the broad application of metabolomics, imaging of metabolites can be challenging due to metabolites&#39; chemical reactivity, structural heterogeneity, and broad concentration range3. However, the concentrations of labile metabolites including high energy compounds, glucose, lactate, glycolytic, pentose shunt pathway, and TCA cycle intermediates, phospholipids, neurotransmitters, signaling compounds, can change within seconds and progress over minutes when tissue enzymes are active during tissue harvesting procedures, such as postmortem ischemia in brain harvesting4,5,6. To ensure accurate metabolomics data acquisition, appropriate and careful sample preparation is critical7,8. Current established platforms for measuring metabolites include NMR, enzyme assays, and mass spectrometry (including liquid and gas chromatography), the last of which is discussed further below.
MALDI-MSI is a state-of-the-art technique that allows for the analysis of complex samples through the detection of individual molecular species. MALDI MSI grants the benefit of being able to quickly and reproducibly measure various molecular compounds in biological samples. Mass spectrometry imaging further allows for the production of images that represent tissue biology based on its composite metabolites, and does so while preserving the spatial distribution of the metabolites in the sample9. MALDI&#39;s ability to detect analytes in a sample without the use of antibody labeling, structural modifications, or other specialized reagents, such as those used in immunostaining, coupled with its ability to monitor hundreds of molecules within a single experiment comprise just a few of the advantages MS imaging grants when it comes to metabolic profiling10,11. In addition to commonly used matrix such as 2,5-dihydroxybenzoic acid (DHB) and 9-aminoacridine (9-AA), recently discovered novel matrix N -(1-Naphthyl) Ethylenediamine Dihydrochloride (NEDC), which is well-suited for analyses of various low molecular weight metabolites, has further improved the application of MALDI MSI in metabolic profiling12.
Despite the broad application of MALDI MSI, the high cost of the instrument and the complexity of the experimental procedure prevents&#160;its wider implementation in individual research laboratories. Therefore, most of the MALDI MSI studies are supported through shared core facilities. The sample preparation, including slide preparation and matrix coating, is the most critical step in MALDI MSI. However, the slide preparation is normally carried out in individual researcher&#39;s laboratory, which creates potential variations in later MALDI MSI acquisition. Here we aim to provide a detailed protocol for the sample preparation of biological samples before proceeding to MALDI MSI measurements, and use a metabolomic profiling of developmental mouse brain as an example.

<table><tbody><tr><td>Andwin Scientific&amp;nbsp;Tissue-Tek CRYO-OCT Compound</td><td>Fisher Scientific&amp;nbsp;</td><td>14-373-65</td><td></td></tr><tr><td>Artist brush MSC #5 1/8 X 9/16 TRIM RED SABLE</td><td>Fisher Scientific&amp;nbsp;</td><td>50-111-2302</td><td></td></tr><tr><td>Autoflex speed MALDI-TOF MS system</td><td>Bruker Daltonics Inc</td><td></td><td>MALDI-TOF MS instrument</td></tr><tr><td>BD&amp;nbsp;Syringe with Luer-Lok Tips</td><td>Fisher Scientific&amp;nbsp;</td><td>14-823-16E</td><td></td></tr><tr><td>BD&amp;nbsp;Vacutainer General Use Syringe Needles</td><td>Fisher Scientific&amp;nbsp;</td><td>23-021-020</td><td></td></tr><tr><td>Bruker Daltonics&amp;nbsp;GLASS SLIDES MALDI IMAGNG</td><td>Fisher Scientific&amp;nbsp;</td><td>NC0380464</td><td></td></tr><tr><td>Drierite, with indicator, 8 mesh, ACROS Organics&amp;nbsp;</td><td>Fisher Scientific</td><td>AC219095000</td><td></td></tr><tr><td>Epson Perfection V600 Photo Scanner</td><td>Amazon</td><td>Perfection V600</td><td></td></tr><tr><td>Fisherbrand&amp;nbsp;5-Place Slide Mailer</td><td>Fisher Scientific&amp;nbsp;</td><td>HS15986</td><td></td></tr><tr><td>Fisherbrand&amp;nbsp;Digital Auto-Range Multimeter</td><td>Fisher Scientific&amp;nbsp;</td><td>01-241-1</td><td></td></tr><tr><td>FlexImaging v3.0</td><td>Bruker Daltonics Inc</td><td></td><td>Bruker MS imaging analysis software</td></tr><tr><td>HPLC Grade Methanol</td><td>Fisher Scientific&amp;nbsp;</td><td>MMX04751</td><td></td></tr><tr><td>HPLC Grade Water</td><td>Fisher Scientific&amp;nbsp;</td><td>W5-1</td><td></td></tr><tr><td>HTX M5 Sprayer</td><td>HTX Technologies, LLC</td><td></td><td>Automatic heated matrix sprayer</td></tr><tr><td>Kimberly-Clark Professional&amp;nbsp;Kimtech Science Kimwipes Delicate Task Wipers</td><td>Fisher Scientific&amp;nbsp;</td><td>06-666A</td><td></td></tr><tr><td>MSC&amp;nbsp;Ziploc Freezer Bag</td><td>Fisher Scientific&amp;nbsp;</td><td>50-111-3769</td><td></td></tr><tr><td>N -(1-Naphthyl) Ethylenediamine Dihydrochloride (NEDC)</td><td>Millipore Sigma Aldrich</td><td>222488</td><td></td></tr><tr><td>SCiLS Lab (2015b)</td><td>SCiLS Lab</td><td></td><td>Advanced MALDI MSI data analysis software</td></tr><tr><td>Thermo Scientific&amp;nbsp;CryoStar NX50 Cryostat</td><td>Fisher Thermo Scientific</td><td>95-713-0</td><td></td></tr><tr><td>Thermo Scientific&amp;nbsp;Nalgene Transparent Polycarbonate Classic Design Desiccator</td><td>Fisher Scientific&amp;nbsp;</td><td>08-642-7</td><td></td></tr></tbody></table>

sample preparation for metabolic profiling using maldi mass spectrometry imaging

Metabolomics, the study to identify and quantify small molecules and metabolites present in an experimental sample, has emerged as an important tool to investigate the biological activities during development and diseases. Metabolomics approaches&#160;are widely employed in the study of cancer, nutrition/diet, diabetes, and other physiological and pathological conditions involving metabolic processes. An advantageous tool that aids in metabolomic profiling advocated in this paper is matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI). Its ability to detect metabolites in situ without labeling, structural modifications, or other specialized reagents, such as those used in immunostaining, makes MALDI MSI a unique tool in advancing methodologies relevant in the field of metabolomics. An appropriate sample preparation process is critical to yield optimal results and will be the focus of this paper.

The representative experiment was performed according to the workflow shown in Figure 1. The developmental C57BL wildtype mouse brains of postnatal day 1, 21, 60 (adult) were harvested as described above following CUNY IACUC guidelines and were snap frozen for 2, 5 and 7 min, respectively, on an aluminum boat floating on liquid nitrogen. The frozen tissues were cryosectioned at 10 &#181;m thickness sections at &#8722;15 &#176;C set for both specimen head and the chamber. The tissue cryosections were then gently transferred onto the pre-cooled conductive side of ITO-coated glass slides for MALDI imaging. Mounted cryosections on ITO slides were desiccated in vacuum for 45 min at room temperature, followed by matrix deposition using automatic matrix sprayer. Matrix NEDC was used to detect metabolites, and a matrix solution of 10 mg/mL in methanol/water (70/30, v/v) was deposited at a flow rate of 0.1 mL/min and a nozzle temperature of 75 &#176;C for 12 cycles with 5 s drying between each cycle. A spray velocity of 1300 mm/min, track spacing of 2 mm, N2 gas pressure of 10 psi and flow rate of 3 L/min and nozzle height of 40 mm was used.
MALDI mass spectra were acquired in negative ion mode by MALDI time of flight (TOF) MSI instrument. 0.5-1 &#181;L of red phosphorus (Pn clusters with n = 1 - 90) emulsion in methanol was deposited onto the ITO slides, next to the mounted tissues, and used to calibrate the instrument in the 100 - 1000 m/z mass range by applying quadratic calibration curve13. The laser spot diameters were focused to &#34;Medium&#34; modulated beam pro&#64257;le for 50 &#181;m raster width. Spectra within the mass range from m/z 50 to 1000 were acquired at a 1000 Hz for 500 shots. Mass spectra data were recorded, and the imaging was further analyzed using advanced MALDI MSI data analysis software. Ion images were generated with root-mean square (RMS) normalization at a bin width of &#177;0.25 Da.
The results in Figure 2 show output images from MALDI MSI data analysis software of m/z spectra selected from every 100 Dalton interval, clearly depicting the utility for identification of spectra from small molecule metabolites to high molecular weight lipids. Each row depicts respective ion heat maps containing both spatial and spectral information of a certain metabolite species across three tissues collected at postnatal day 1, 21, and 60. For each representative m/z, analysis of regional distribution and ion abundances can be used to compare relative amounts of corresponding species between different ages. A strength of the MALDI MSI methodology is the ability to discern the specificity of certain species identified by m/z to developmental milestones or specific anatomical structures. Some metabolites are observed to be enriched in P1 neonates (m/z 320.1), enriched in P60 adults (m/z 846.5), or uniformly distributed across ages (m/z 480.3); other molecular species are specifically enriched in gray matter (m/z 117.1; 524.3; 765.1), white matter (m/z 673.4; 846.5), or CSF/ventricles (m/z 239.0) (Figure 2A). The spatial distribution of representative metabolites including hypoxanthine (m/z 135.0),&#160;N-Acetyl-L-aspartic acid (m/z 174.0), Arachidonic acid (m/z 303.2), and several lipids such as lysophosphatidylethanolamine LPE (18:1) (m/z 478.3), LPE(20:4) (m/z 500.3), LPE (22:6) (m/z 524.3), phosphatidylethanolamine PE (44:10) (m/z 838.5), Phosphatidylinositol PI(38:4) (m/z 885.6) are also shown (Figure&#160;2B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62008/62008fig01.jpg" /> 
Figure 1. Workflow of MALDI- time of flight (TOF) mass spectrometry imaging. The snap frozen tissue is cryrosectioned in cryostat and mounted on ITO slide. &#8594; The slide with tissue sections is coated with a fine layer of matrix using automatic matrix sprayer. &#8594;&#160;Mass spectra is collected by MALDI-TOF MSI instrument at a raster of 20-200um. &#8594;&#160;The data is analyzed, and the images are generated using advanced MALDI MSI data analysis software. Scale bar: 2 mm. <a href="https://www.jove.com/files/ftp_upload/62008/62008fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62008/62008fig2v2.jpg" /> 
Figure 2. Representative output with selected m/z spectra from mass spectrometry acquired at 50 &#181;m lateral resolution. (A) A heat map depicts the spatial distribution of specific metabolite species selected from every 100 Dalton m/z interval across the three developmental milestones identified at P1, P21 and P60. (B) The spatial distribution of representative metabolites at P1, P21 and P60, including: hypoxanthine,&#160;N-Acetyl-L-aspartic acid, Arachidonic acid, and different lipid species such as lysophosphatidylethanolamine (LPE), phosphatidylethanolamine (PE), Phosphatidylinositol (PI). Scale bar, 500 &#181;m.&#160;<a href="https://www.jove.com/files/ftp_upload/62008/62008fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Sample Preparation for Metabolic Profiling using MALDI Mass Spectrometry Imaging at JoVE.com

Sample Preparation for Metabolic Profiling using MALDI Mass Spectrometry Imaging

The goal of this protocol is to provide detailed guidance on the sample preparation when planning for experiments using MALDI MSI to maximize metabolic and molecular detection in biological samples.

Metabolomics, the study to identify and quantify small molecules and metabolites present in an experimental sample, has emerged as an important tool to ...

sample-preparation-for-metabolic-profiling-using-maldi-mass

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Biology

6.0K Views.  The City University of New York. Het doel van dit protocol is om gedetailleerde richtlijnen te geven over de monstervoorbereiding bij het plannen van experimenten met MALDI MSI om metabole en moleculaire detectie in biologische monsters te maximaliseren. 

Video: Sample Preparation for Metabolic Profiling using MALDI Mass Spectrometry Imaging

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

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

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

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

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

Chemistry

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.1-3 The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.4,5 Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.6 Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.7-9 Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples. 
Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.10,11 Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.

We developed a technology based on mesoporous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. The physico-chemical properties of our mesoporous chips were finely tuned to provide substantial control in peptide enrichment and consequently profile the serum proteome for diagnostic purposes.

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Bioengineering

Optimal Preparation of Formalin Fixed Samples for Peptide Based Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Workflows

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host of biomolecules from drugs and lipids to N-glycans. Although various sample preparation techniques exist, detecting peptides from formaldehyde preserved tissues remains one of the most difficult challenges for this type of mass spectrometric analysis. For this reason, we have created and optimized a robust methodology that preserves the spatial information contained within the sample, while eliciting the greatest number of ionizable peptides. We have also aimed to achieve this in a cost effective and simple way, thereby eliminating potential bias or preparation error, which can occur when using automated instrumentation. The end result is a reproducible and inexpensive protocol.

This protocol describes a reproducible and reliable method for the sublimation-based preparation of formalin fixed tissue destined for imaging mass spectrometry.

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Biochemistry

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein-protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein-metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our protocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass spectrometry (MS)-based protein and metabolite detection. In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS or UPLC-MS/MS). Empty-vector (EV) control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in parallel in near-physiological conditions (cellular lysate). The presented method is straightforward, fast, and can be easily adapted to biological systems other than plant cell cultures.

Protein-protein and protein-metabolite interactions are crucial for all cellular functions. Herein, we describe a protocol that allows parallel analysis of these interactions with a protein of choice. Our protocol was optimized for plant cell cultures and combines affinity purification with mass spectrometry-based protein and metabolite detection.

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

Single-throughput Complementary High-resolution Analytical Techniques for Characterizing Complex Natural Organic Matter Mixtures

Natural organic matter (NOM) is composed of a highly complex mixture of thousands of organic compounds which, historically, proved difficult to characterize. However, to understand the thermodynamic and kinetic controls on greenhouse gas (carbon dioxide [CO2] and methane [CH4]) production resulting from the decomposition of NOM, a molecular-level characterization coupled with microbial proteome analyses is necessary. Further, climate and environmental changes are expected to perturb natural ecosystems, potentially upsetting complex interactions that influence both the supply of organic matter substrates and the microorganisms performing the transformations. A detailed molecular characterization of the organic matter, microbial proteomics, and the pathways and transformations by which organic matter is decomposed will be necessary to predict the direction and magnitude of the effects of environmental changes. This article describes a methodological throughput for comprehensive metabolite characterization in a single sample by direct injection Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), gas chromatography mass spectrometry (GC-MS), nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography mass spectrometry (LC-MS), and proteomics analysis. This approach results in a fully-paired dataset which improves statistical confidence for inferring pathways of organic matter decomposition, the resulting CO2 and CH4 production rates, and their responses to environmental perturbation. Herein we present results of applying this method to NOM samples collected from peatlands; however, the protocol is applicable to any NOM sample (e.g., peat, forested soils, marine sediments, etc.).

This protocol describes a single throughput for complementary analytical and omics techniques culminating in a fully-paired characterization of natural organic matter and microbial proteomics in different ecosystems. This approach permits robust comparisons for identifying metabolic pathways and transformations important for describing greenhouse gas production and predicting responses to environmental change.

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Environment

Using the Open-Source MALDI TOF-MS IDBac Pipeline for Analysis of Microbial Protein and Specialized Metabolite Data

In order to visualize the relationship between bacterial phylogeny and specialized metabolite production of bacterial colonies growing on nutrient agar, we developed IDBac&#8212;a low-cost and high-throughput matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) bioinformatics pipeline. IDBac software is designed for non-experts, is freely available, and capable of analyzing a few to thousands of bacterial colonies. Here, we present procedures for the preparation of bacterial colonies for MALDI-TOF MS analysis, MS instrument operation, and data processing and visualization in IDBac. In particular, we instruct users how to cluster bacteria into dendrograms based on protein MS fingerprints and interactively create Metabolite Association Networks (MANs) from specialized metabolite data.

IDBac is an open-source mass spectrometry-based bioinformatics pipeline that integrates data from both intact protein and specialized metabolite spectra, collected on cell material scraped from bacterial colonies. The pipeline allows researchers to rapidly organize hundreds to thousands of bacterial colonies into putative taxonomic groups, and further differentiate them based on specialized metabolite production.

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Multi-step Preparation Technique to Recover Multiple Metabolite Compound Classes for In-depth and Informative Metabolomic Analysis

Metabolomics is an emerging field which enables profiling of samples from living organisms in order to obtain insight into biological processes. A vital aspect of metabolomics is sample preparation whereby inconsistent techniques generate unreliable results. This technique encompasses protein precipitation, liquid-liquid extraction, and solid-phase extraction as a means of fractionating metabolites into four distinct classes.&#160;Improved enrichment of low abundance molecules with a resulting increase in sensitivity is obtained, and ultimately results in more confident identification of molecules. This technique has been applied to plasma, bronchoalveolar lavage fluid, and cerebrospinal fluid samples with volumes as low as 50 &#181;l. &#160;Samples can be used for multiple downstream applications; for example, the pellet resulting from protein precipitation can be stored for later analysis. The supernatant from that step undergoes liquid-liquid extraction using water and strong organic solvent to separate the hydrophilic and hydrophobic compounds. Once fractionated, the hydrophilic layer can be processed for later analysis or discarded if not needed. The hydrophobic fraction is further treated with a series of solvents during three solid-phase extraction steps to separate it into fatty acids, neutral lipids, and phospholipids. This allows the technician the flexibility to choose which class of compounds is preferred for analysis. It also aids in more reliable metabolite identification since some knowledge of chemical class exists.

The reliability of results in metabolomics experiments depends on the effectiveness and reproducibility of the sample preparation. Described is a rigorous and in-depth method that enables extraction of metabolites from biological fluids with the option of subsequently analyzing up to thousands of compounds, or just the compound classes of interest.

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Preparation of Drosophila Larval Samples for Gas Chromatography-Mass Spectrometry (GC-MS)-based Metabolomics

Recent advances in the field of metabolomics have established the fruit fly Drosophila melanogaster as a powerful genetic model for studying animal metabolism. By combining the vast array of Drosophila genetic tools with the ability to survey large swaths of intermediary metabolism, a metabolomics approach can reveal complex interactions between diet, genotype, life-history events, and environmental cues. In addition, metabolomics studies can discover novel enzymatic mechanisms and uncover previously unknown connections between seemingly disparate metabolic pathways. In order to facilitate more widespread use of this technology among the Drosophila community, here we provide a detailed protocol that describes how to prepare Drosophila larval samples for gas chromatography-mass spectrometry (GC-MS)-based metabolomic analysis. Our protocol includes descriptions of larval sample collection, metabolite extraction, chemical derivatization, and GC-MS analysis. Successful completion of this protocol will allow users to measure the relative abundance of small polar metabolites, including amino acids, sugars, and organic acids involved in glycolysis and the TCA cycles.

Preparation of Drosophila Larval Samples for Gas Chromatography-Mass Spectrometry GC-MS-based Metabolomics

This protocol describes how to prepare Drosophila larvae for GC-MS-based metabolomic analysis.

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Developmental Biology

Dithranol as a Matrix for Matrix Assisted Laser Desorption/Ionization Imaging on a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer

Mass spectrometry imaging (MSI) determines the spatial localization and distribution patterns of compounds on the surface of a tissue section, mainly using MALDI (matrix&#160;assisted laser desorption/ionization)-based analytical techniques. New matrices for small-molecule MSI, which can improve the analysis of low-molecular weight (MW) compounds, are needed. These matrices should provide increased analyte signals while decreasing MALDI background signals. In addition, the use of ultrahigh-resolution instruments, such as Fourier transform ion cyclotron resonance (FTICR) mass spectrometers, has&#160;the ability to resolve analyte signals from matrix signals, and this can partially overcome many problems associated with the background originating from the MALDI matrix. The reduction in the intensities of the metastable matrix clusters by FTICR MS can also help to overcome some of the interferences associated with matrix peaks on other instruments. High-resolution instruments such as the FTICR mass spectrometers are advantageous as they can produce distribution patterns of many compounds simultaneously while still providing confidence in chemical identifications. Dithranol (DT; 1,8-dihydroxy-9,10-dihydroanthracen-9-one) has previously been reported as a MALDI matrix for tissue imaging. In this work, a protocol for the use of DT for MALDI imaging of endogenous lipids from the surfaces of mammalian tissue sections, by positive-ion MALDI-MS, on an ultrahigh-resolution hybrid quadrupole FTICR instrument has been provided.

Dithranol (DT; 1,8-dihydroxy-9,10-dihydroanthracen-9-one) has previously been reported as a MALDI matrix for tissue imaging of small molecules; protocols for the use of DT for the MALDI imaging of endogenous lipids on the surface of tissue sections&#160;by positive-ion MALDI-MS on an ultrahigh-resolution quadrupole-FTICR instrument are provided here.

Mass spectrometry imaging (MSI) determines the spatial localization and distribution patterns of compounds on the surface of a tissue section, mainly ...

A Strategy for Sensitive, Large Scale Quantitative Metabolomics

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting factors, such as labor intensive sample preparations, low detection limits, slow scan speeds, intensive method optimization for each metabolite, and the inability to measure both positively and negatively charged ions in single experiments. Therefore, a novel metabolomics protocol could advance metabolomics studies. Amide-based hydrophilic chromatography enables polar metabolite analysis without any chemical derivatization. High resolution MS using the Q-Exactive (QE-MS) has improved ion optics, increased scan speeds (256 msec at resolution 70,000), and has the capability of carrying out positive/negative switching. Using a cold methanol extraction strategy, and coupling an amide column with QE-MS enables robust detection of 168 targeted polar metabolites and thousands of additional features simultaneously.&#160; Data processing is carried out with commercially available software in a highly efficient way, and unknown features extracted from the mass spectra can be queried in databases.

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. Utilizing normal-phased liquid chromatography coupled to high-resolution mass spectrometry with polarity switching and a rapid duty cycle, we describe a protocol to analyze the polar metabolic composition of biological material with high sensitivity, accuracy, and resolution.

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting ...

Sample Preparation for Metabolic Profiling using MALDI Mass Spectrometry Imaging

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