Services and support of the research project were provided by the VCU Massey Cancer Center Tissue and Data Acquisition and Analysis Core and the VCU Lipidomics and Metabolomics Core, which are supported in part with funding from NIH-NCI Cancer Center Support Grant P30CA016059. This work was supported by National Institutes of Health Grants R21CA232234 (Santiago Lima).

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	<li>Maceyka, M., Spiegel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sphingolipid+metabolites+in+inflammatory+disease.">Sphingolipid metabolites in inflammatory disease.</a> Nature. 510, 58-67 (2014).</li><li>Ogretmen, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sphingolipid+metabolism+in+cancer+signalling+and+therapy.">Sphingolipid metabolism in cancer signalling and therapy.</a> Nature Reviews. Cancer. 18 (1), 33-50 (2018).</li><li>Hannun, Y. A., Obeid, L. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sphingolipid+signaling+in+metabolic+disorders.">Sphingolipid signaling in metabolic disorders.</a> Cell Metabolism. 16 (4), 420-434 (2012).</li><li>Lima, S., Milstien, S., Spiegel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sphingosine+and+Sphingosine+Kinase+1+involvement+in+endocytic+membrane+Trafficking.">Sphingosine and Sphingosine Kinase 1 involvement in endocytic membrane Trafficking.</a> The Journal of Biological Chemistry. 292 (8), 3074-3088 (2017).</li><li>Lima, S., 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=TP53+is+required+for+BECN1-+and+ATG5-dependent+cell+death+induced+by+sphingosine+kinase+1+inhibition.">TP53 is required for BECN1- and ATG5-dependent cell death induced by sphingosine kinase 1 inhibition.</a> Autophagy. , 1-50 (2018).</li><li>Young, M. 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=Sphingosine+Kinase+1+cooperates+with+autophagy+to+maintain+endocytic+membrane+trafficking.">Sphingosine Kinase 1 cooperates with autophagy to maintain endocytic membrane trafficking.</a> Cell Reports. 17 (6), 1532-1545 (2016).</li><li>Young, M. M., Wang, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sphingolipids+as+regulators+of+autophagy+and+endocytic+trafficking.">Sphingolipids as regulators of autophagy and endocytic trafficking.</a> Advances in Cancer Research. 140, 27-60 (2018).</li><li>Shen, H., 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=Coupling+between+endocytosis+and+sphingosine+kinase+1+recruitment.">Coupling between endocytosis and sphingosine kinase 1 recruitment.</a> Nature Cell Biology. 16 (7), 652-662 (2014).</li><li>Morad, S. A. F., Cabot, M. C., Chalfant, C. E., Fisher, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Advances in Cancer Research. 140, 235-263 (2018).</li><li>Rohrbach, T. D., 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=A+simple+method+for+sphingolipid+analysis+of+tissues+embedded+in+optimal+cutting+temperature+compound.">A simple method for sphingolipid analysis of tissues embedded in optimal cutting temperature compound.</a> Journal of Lipid Research. 61 (6), 953-967 (2020).</li><li>Weston, L. A., Hummon, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+LC-MS/MS+analysis+of+optimal+cutting+temperature+(OCT)+compound+removal+for+the+study+of+mammalian+proteomes.">Comparative LC-MS/MS analysis of optimal cutting temperature (OCT) compound removal for the study of mammalian proteomes.</a> Analyst. 138 (21), 6380-6384 (2013).</li><li>Holfeld, A., Vald&#233;s, A., Malmstr&#246;m, P. -. U., Segersten, U., Lind, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+proteomic+workflow+for+mass+spectrometric+analysis+of+tissue+samples+preserved+by+different+methods.">Parallel proteomic workflow for mass spectrometric analysis of tissue samples preserved by different methods.</a> Analytical Chemistry. 90 (9), 5841-5849 (2018).</li><li>Zhang, W., Sakashita, S., Taylor, P., Tsao, M. S., Moran, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+proteome+analysis+of+fresh+frozen+and+optimal+cutting+temperature+(OCT)+embedded+primary+non-small+cell+lung+carcinoma+by+LC-MS/MS.">Comprehensive proteome analysis of fresh frozen and optimal cutting temperature (OCT) embedded primary non-small cell lung carcinoma by LC-MS/MS.</a> Methods. 81, 50-55 (2015).</li><li>Shah, P., 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=Tissue+proteomics+using+chemical+immobilization+and+mass+spectrometry.">Tissue proteomics using chemical immobilization and mass spectrometry.</a> Analytical Biochemistry. 469, 27-33 (2015).</li></ol>

The authors have nothing to disclose.

OCT is a common long-term cryo-preservation agent used in biorepositories. However, OCT can result in ion suppression when tissues are analyzed by various mass spectrometry platforms12,13,14,15, or result in loss of signal when samples are analyzed by LC-ESI-MS/MS11. OCT in cryopreserved tissues can also interfere with tissue normalization methods such as weighing and pr...

Sphingolipids are bioactive metabolites known for their roles in human metabolism and disease1,2. They regulate complex cellular processes such as cell migration, cell survival and death, cell movement, vesicular trafficking, cellular invasion and metastasis, angiogenesis, and the production of cytokines1,2,3,4,5,6,7,8,9. Defects in the regulation of sphingolipid metabolism contribute to the initiation and progression of cancers, determine how aggressive cancers are, and how cancers respond to and develop resistance to therapy3,10. Therefore, because of these broad impacts on the etiology of disease, analytical methods that can precisely establish disease-specific sphingolipid alterations are important tools. Mass spectrometry (MS) is the most accurate and reliable method to analyze sphingolipid alterations.
Human specimens that can be used for the analysis of sphingolipid alterations can be obtained prospectively from the surgical suite or retrospectively from tissue biorepositories. Fresh tissues from surgery are advantageous because they can be directly analyzed by MS or other analytical methods. However, acquiring tissues prospectively has administrative, technical, and logistical hurdles, and collecting sufficient specimens to reach statistical power can be challenging. Obtaining tissues from biorepositories is advantageous because they can be acquired retrospectively, in large numbers, and biorepositories confirm histology and pathology, use standard operating procedures to cryogenically preserve and store tissues, and can provide clinicopathological data that can be used for correlation analyses. However, to preserve molecular and structural features, biorepositories may cryopreserve tissues by embedding them in optimal cutting temperature (OCT) compound, which we have shown interferes with data normalization assays and the quantification of sphingolipids by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS)11. It has also been shown that polyvinyl alcohol and polyethylene glycol, the primary components in OCT compound, result in ion suppression in other MS analysis platforms12,13,14,15. Therefore, OCT compound must be removed from tissues prior to sphingolipidomic analysis by MS.
In a previous report, we have validated a protocol for the removal of OCT compound from human specimens for LC-ESI-MS/MS analysis11 and the methodology used for weighing tissues for data normalization11. Here, we detail the steps of the sphingolipidomic OCT compound-removal protocol (sOCTrP) and show representative data from human lung adenocarcinoma tumors and normal adjacent uninvolved tissues.

<table><tbody><tr><td>1 mL polypropylene pipette tips</td><td>NA</td><td>NA</td><td>Used to retrieve specimens</td></tr><tr><td>1.5 mL polypropylene centrifuge tubes</td><td>NA</td><td>NA</td><td></td></tr><tr><td>10 mL Erlenmeyer flask</td><td>VWR</td><td>89091-116</td><td>Used for tube and tissue weighing</td></tr><tr><td>AB Sciex Analyst 1.6.2</td><td>Sciex</td><td>NA</td><td>Software to analyze and integrate MS data</td></tr><tr><td>Ammonium formate</td><td>Fisher Scientific</td><td>A11550</td><td>For LC mobile phases</td></tr><tr><td>Analytical scale</td><td>NA</td><td>NA</td><td>Scale that is accurate to 0.1 mg</td></tr><tr><td>Bottle top dispenser</td><td>Sartorius</td><td>LH-723071</td><td>Used for dispensing solvents</td></tr><tr><td>C12-Ceramide (d18:1/C12:0); N-(dodecanoyl)-sphing-4-enine</td><td>Avanti Polar Lipids</td><td>LM2212</td><td>Internal standard</td></tr><tr><td>C12-glucosylceramide (d18:1/12:0); N-(dodecanoyl)-1-&amp;beta;-glucosyl-sphing-4-eine</td><td>Avanti Polar Lipids</td><td>LM2511</td><td>Internal standard</td></tr><tr><td>C12-lactosylceramide (d18:1/12:0); N-(dodecanoyl)-1-&amp;szlig;-lactosyl-sphing-4-ene</td><td>Avanti Polar Lipids</td><td>LM2512</td><td>Internal standard</td></tr><tr><td>C12-Sphingomyelin&amp;nbsp; (d18:1/C12:0), N-(dodecanoyl)-sphing-4-enine-1-phosphocholine</td><td>Avanti Polar Lipids</td><td>LM2312</td><td>Internal standard</td></tr><tr><td>CHLOROFORM OMNISOLV 4L</td><td>VWR</td><td>EM-CX1054-1</td><td></td></tr><tr><td>ClickSeal Biocontainment Lids</td><td>Thermo Scientific</td><td>75007309</td><td>To prevent biohazard aeresols during centrifugation</td></tr><tr><td>Conflikt</td><td>Decon Labs</td><td>4101</td><td>Decontaminant</td></tr><tr><td>CTO-20A/20AC Column Oven</td><td>Shimadzu</td><td>NA</td><td>For LC</td></tr><tr><td>d17:1-Sphingosine;&amp;nbsp; (2S,3R,4E)-2-aminoheptadec-4-ene-1,3-diol</td><td>Avanti Polar Lipids</td><td>LM2000</td><td>Internal standard</td></tr><tr><td>d17:1-Sphingosine-1-phosphate; heptadecasphing-4-enine-1-phosphate</td><td>Avanti Polar Lipids</td><td>LM2144</td><td>Internal standard</td></tr><tr><td>DGU20A5R degasser</td><td>Shimadzu</td><td>NA</td><td></td></tr><tr><td>Disposable Culture Tubes 13x100mm</td><td>VWR</td><td>53283-800</td><td>13x100 mm screw top tubes</td></tr><tr><td>Heated water bath</td><td>NA</td><td>NA</td><td>For overnight lipid extraction</td></tr><tr><td>Homogenizer 150</td><td>Fisher Scientific</td><td>15-340-167</td><td>triturate tissues</td></tr><tr><td>Homogenizer Plastic Disposable Generator Probe</td><td>Fisher Scientific</td><td>15-340-177</td><td>for homogenization</td></tr><tr><td>Kimwipes</td><td>Kimtech</td><td>34120</td><td>Laboratory grade tissue used to make wicks</td></tr><tr><td>Methanol LC-MS Grade 4L</td><td>VWR</td><td>EM-MX0486-1</td><td></td></tr><tr><td>Nexera LC-30 AD binary pump system</td><td>Shimadzu</td><td>NA</td><td>For LC-MS</td></tr><tr><td>Permanent marker</td><td>VWR</td><td>52877-310</td><td></td></tr><tr><td>Phenolic Screw Thread Closure, Kimble Chase (caps for disposable culture tubes)</td><td>VWR</td><td>89001-502</td><td>13x100 mm screw top tube caps</td></tr><tr><td>Phosphate bufffered saline</td><td>Thermo Scientific</td><td>10010023</td><td>To retrieve specimens from tubes after washing</td></tr><tr><td>Repeater pipette</td><td>Eppendorf</td><td>4987000118</td><td>To dispense LC-MS internal standards</td></tr><tr><td>Screw Caps, Blue, Red PTFE/White Silicone</td><td>VWR</td><td>89239-020</td><td>Autoinjector vial caps</td></tr><tr><td>Screw Thread Glass Vials with ID Patch</td><td>VWR</td><td>46610-724</td><td>Autoinjector vials</td></tr><tr><td>SIL-30AC autoinjector</td><td>Shimadzu</td><td>NA</td><td></td></tr><tr><td>SpeedVac</td><td>Thermo Scientific</td><td>SPD2030P1220</td><td>For drying solvents</td></tr><tr><td>Supelco 2.1 (i.d.) x 50 mm Ascentis Express C18 column</td><td>Sigma Aldrich</td><td>581300-U</td><td>For LC-MS</td></tr><tr><td>Triple Quad 5500+ LC-MS/MS System</td><td>Sciex</td><td>NA</td><td>For LC-ESI-MS/MS</td></tr><tr><td>Ultrasonic water bath</td><td>Branson</td><td>Model 2800</td><td>for homogenization and resuspension of extracted and dried lipids</td></tr><tr><td>Vortexer</td><td>NA</td><td>NA</td><td>For sOCTrP and resuspending dried lipids</td></tr><tr><td>VWR Culture Tubes Disposable Borosilicate Glass</td><td>VWR</td><td>47729572</td><td>13x100 glass culture tubes</td></tr><tr><td>Water Hipersolve Chromanorm LC-MS</td><td>VWR</td><td>BDH83645.400</td><td></td></tr></tbody></table>

preparation of human tissues embedded in optimal cutting temperature compound for mass spectrometry analysis

Sphingolipids are cellular components that have well-established roles in human metabolism and disease. Mass spectrometry can be used to determine whether sphingolipids are altered in a disease and investigate whether sphingolipids can be targeted clinically. However, properly powered prospective studies that acquire tissues directly from the surgical suite can be time consuming, and technically, logistically, and administratively challenging. In contrast, retrospective studies can take advantage of cryopreserved human specimens already available, usually in large numbers, at tissue biorepositories. Other advantages of procuring tissues from biorepositories include access to information associated with the tissue specimens including histology, pathology, and in some instances clinicopathological variables, all of which can be used to examine correlations with lipidomics data. However, technical limitations related to the incompatibility of optimal cutting temperature compound (OCT) used in the cryopreservation and mass spectrometry is a technical barrier for the analysis of lipids. However, we have previously shown that OCT can be easily removed from human biorepository specimens through cycles of washes and centrifugation without altering their sphingolipid content. We have also previously established that sphingolipids in human tissues cryopreserved in OCT are stable for up to 16 years. In this report, we outline the steps and workflow to analyze sphingolipids in human tissue specimens that are embedded in OCT, including washing tissues, weighing tissues for data normalization, the extraction of lipids, preparation of samples for analysis by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS), mass spectrometry data integration, data normalization, and data analysis.

In this protocol, we describe in detail a method to remove OCT from cryo-preserved human tissues and weigh the tissues for analysis by LC-ESI-MS/MS. The materials required for this procedure are listed in Table of Materials. Shown in Figure 1 are results of a typical experiment where 10 human lung adenocarcinoma tumors and 10 normal adjacent tissues were washed to remove OCT and analyzed by LC-ESI-MS/MS. Importantly, as we have previously shown11, the...

Watch this Scientific Journal Video about Preparation of Human Tissues Embedded in Optimal Cutting Temperature Compound for Mass Spectrometry Analysis at JoVE.com

Preparation of Human Tissues Embedded in Optimal Cutting Temperature Compound for Mass Spectrometry Analysis

Sphingolipids are bioactive metabolites with well-established roles in human disease. Characterizing alterations in tissues with mass-spectrometry can reveal roles in disease etiology or identify therapeutic targets. However, the OCT-compound used for cryopreservation in biorepositories interferes with mass-spectrometry. We outline methods to analyze sphingolipids in human tissues embedded in OCT with LC-ESI-MS/MS.

Sphingolipids are cellular components that have well-established roles in human metabolism and disease. Mass spectrometry can be used to determine whether ...

preparation-human-tissues-embedded-optimal-cutting-temperature

Research

JoVE Journal

Cancer Research

2.0K Views.  Virginia Commonwealth University. OCT is een cryoconserveringsmiddel dat niet compatibel is met massaspectrometrie-analyse. Met behulp van dit protocol kunnen onderzoekers OCT uit weefsels verwijderen en sfingolipiden kwantificeren met tandemvloeistofchromatografie massaspectrometrie. De voorbereiding van weefseldrogende lonten, weefselwassen en weefselweging zal worden gedemonstreerd door April Boyd van de Virginia Commonwealth University, en de lipide-extractie zal worden gedemonstreerd door Dr. Jeremy Allegood, directeur v...

Video: Preparation of Human Tissues Embedded in Optimal Cutting Temperature Compound for Mass Spectrometry Analysis

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.

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host ...

Biochemistry

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 ...

Bioengineering

Sample Preparation for Probe Electrospray Ionization Mass Spectrometry

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ratios (m/z), which are useful to deduce molecular weights and structures. While it is essentially a destructive analytical method, recent advancements in the ambient ionization technique have enabled us to acquire data while leaving tissue in a relatively intact state in terms of integrity. Probe electrospray ionization (PESI) is a so-called direct method because it does not require complex and time-consuming pretreatment of samples. A fine needle serves as a sample picker, as well as an ionization emitter. Based on the very sharp and fine property of the probe tip, destruction of the samples is minimal, allowing us to acquire the real-time molecular information from living things in situ. Herein, we introduce three applications of PESI-MS technique that will be useful for biomedical research and development. One involves the application to solid tissue, which is the basic application of this technique for the medical diagnosis. As this technique requires only 10 mg of the sample, it may be very useful in the routine clinical settings. The second application is for in vitro medical diagnostics where human blood serum is measured. The ability to measure fluid samples is also valuable in various biological experiments where a sufficient volume of sample for conventional analytical techniques cannot be provided. The third application leans toward the direct application of probe needles in living animals, where we can obtain real-time dynamics of metabolites or drugs in specific organs. In each application, we can infer the molecules that have been detected by MS or use artificial intelligence to obtain a medical diagnosis.

This article introduces sample preparation methods for a unique real-time analytical method based on the ambient mass spectrometry. This method lets us perform real-time analysis of the biological molecules in vivo without any special pretreatments.

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ...

Expanding the Comprehension of the Tumor Microenvironment using Mass Spectrometry Imaging of Formalin-Fixed and Paraffin-Embedded Tissue Samples

Advances in immune-based therapies have revolutionized cancer treatment and research. This has triggered growing demand for the characterization of the tumor immune landscape. Although standard immunohistochemistry is suitable for studying tissue architecture, it is limited to the analysis of a small number of markers. Conversely, techniques such as flow cytometry can evaluate multiple markers simultaneously, although information about tissue morphology is lost. In recent years, multiplexed strategies that integrate phenotypic and spatial analysis have emerged as comprehensive approaches to the characterization of the tumor immune landscape. Herein, we discuss an innovative technology combining metal-labeled antibodies and secondary ion mass spectrometry focusing on the technical steps in assay development and optimization, tissue preparation, and image acquisition and processing. Before staining, a metal-labeled antibody panel must be developed and optimized. This hi-plex image system supports up to 40 metal-tagged antibodies in a single tissue section. Of note, the risk of signal interference increases with the number of markers included in the panel. After panel design, particular attention should be given to the metal isotope assignment to the antibody to minimize this interference. Preliminary panel testing is performed using a small subset of antibodies and subsequent testing of the entire panel in control tissues. Formalin-fixed, paraffin-embedded tissue sections are obtained and mounted on gold-coated slides and further stained. The staining takes 2 days and closely resembles standard immunohistochemical staining. Once samples are stained, they are placed in the image acquisition instrument. Fields of view are selected, and images are acquired, uploaded, and stored. The final stage is image preparation for the filtering and removal of interference using the system&#39;s image processing software. A disadvantage of this platform is the lack of analytical software. However, the images generated are supported by different computational pathology software.

In the era of cancer immunotherapy, interest in elucidating tumor microenvironment dynamics has increased strikingly. This protocol details a mass spectrometry imaging technique with respect to its staining and imaging steps, which allow for highly multiplexed spatial analysis.

Advances in immune-based therapies have revolutionized cancer treatment and research. This has triggered growing demand for the characterization of the ...

Proteomic Sample Preparation from Formalin Fixed and Paraffin Embedded Tissue

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale quantitative analysis of formalin fixed and paraffin embedded (FFPE) tissue. The procedure comprises four distinct steps. The first one is the preparation of sections from the FFPE material and microdissection of cells of interest. In the second step the isolated cells are lysed and processed using 'filter aided sample preparation' (FASP) technique. In this step, proteins are depleted from reagents used for the sample lysis and are digested in two-steps using endoproteinase LysC and trypsin. 
After each digestion, the peptides are collected in separate fractions and their content is determined using a highly sensitive fluorescence measurement. Finally, the peptides are fractionated on 'pipette-tip' microcolumns. The LysC-peptides are separated into 4 fractions whereas the tryptic peptides are separated into 2 fractions. In this way prepared samples allow analysis of proteomes from minute amounts of material to a depth of 10,000 proteins. Thus, the described workflow is a powerful technique for studying diseases in a system-wide-fashion as well as for identification of potential biomarkers and drug targets.

Archival formalin fixed and paraffin embedded (FFPE) clinical samples are valuable material for investigation of diseases. Here we demonstrate a sample preparation workflow allowing in-depth proteomic analysis of microdissected FFPE tissue.

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale ...

Biology

Isolation and Profiling of Human Primary Mesenteric Arterial Endothelial Cells at the Transcriptome Level

Endothelial cells (ECs) are crucial for vascular and whole-body function through their dynamic response to environmental cues. Elucidating the transcriptome and epigenome of ECs is paramount to understanding their roles in development, health, and disease, but is limited in the availability of isolated primary cells. Recent technologies have enabled the high-throughput profiling of EC transcriptome and epigenome, leading to the identification of previously unknown EC cell subpopulations and developmental trajectories. While EC cultures are a useful tool in the exploration of EC function and dysfunction, the culture conditions and multiple passages can introduce external variables that alter the properties of native EC, including morphology, epigenetic state, and gene expression program. To overcome this limitation, the present paper demonstrates a method of isolating human primary ECs from donor mesenteric arteries aiming to capture their native state. ECs in the intimal layer are dissociated mechanically and biochemically with the use of particular enzymes. The resultant cells can be directly used for bulk RNA or single-cell RNA-sequencing or plated for culture. In addition, a workflow is described for the preparation of human arterial tissue for spatial transcriptomics, specifically for a commercially available platform, although this method is also suitable for other spatial transcriptome profiling techniques. This methodology can be applied to different vessels collected from a variety of donors in health or disease states to gain insights into EC transcriptional and epigenetic regulation, a pivotal aspect of endothelial cell biology.

The protocol describes the isolation, culture, and profiling of endothelial cells from human mesenteric artery. Additionally, a method is provided to prepare human artery for spatial transcriptomics. Proteomics, transcriptomics, and functional assays can be performed on isolated cells. This protocol can be repurposed for any medium- or large-size artery.

Endothelial cells (ECs) are crucial for vascular and whole-body function through their dynamic response to environmental cues. Elucidating the ...

Medicine

High-Throughput, Multi-Image Cryohistology of Mineralized Tissues

There is an increasing need for efficient phenotyping and histopathology of a variety of tissues. This phenotyping need is evident with the ambitious projects to disrupt every gene in the mouse genome. The research community needs rapid and inexpensive means to phenotype tissues via histology. Histological analyses of skeletal tissues are often time consuming and semi-quantitative at best, regularly requiring subjective interpretation of slides from trained individuals. Here, we present a cryohistological paradigm for efficient and inexpensive phenotyping of mineralized tissues. First, we present a novel method of tape-stabilized cryosectioning that preserves the morphology of mineralized tissues. These sections are then adhered rigidly to glass slides and imaged repeatedly over several rounds of staining. The resultant images are then aligned either manually or via computer software to yield composite stacks of several layered images. The protocol allows for co-localization of numerous molecular signals to specific cells within a given section. In addition, these fluorescent signals can be quantified objectively via computer software. This protocol overcomes many of the shortcomings associated with histology of mineralized tissues and can serve as a platform for high-throughput, high-content phenotyping of musculoskeletal tissues moving forward.

In this manuscript, we present a high-throughput, semi-automated cryohistology platform to produce aligned composite images of multiple response measures from several rounds of fluorescent imaging on frozen sections of mineralized tissues.

There is an increasing need for efficient phenotyping and histopathology of a variety of tissues. This phenotyping need is evident with the ambitious ...

High-Resolution 3D Reconstruction of Human Brain with SHORT and LSFM

Optical Clearing and Labeling for Light-sheet Fluorescence Microscopy in Large-scale Human Brain Imaging

Despite the numerous clearing techniques that emerged in the last decade, processing postmortem human brains remains a challenging task due to its dimensions and complexity, which make imaging with micrometer resolution particularly difficult. This paper presents a protocol to perform the reconstruction of volumetric portions of the human brain by simultaneously processing tens of sections with the SHORT (SWITCH - H2O2 - Antigen Retrieval - 2,2'-thiodiethanol [TDE]) tissue transformation protocol, which enables clearing, labeling, and sequential imaging of the samples with light-sheet fluorescence microscopy (LSFM). SHORT provides rapid tissue clearing and homogeneous multi-labeling of thick slices with several neuronal markers, enabling the identification of different neuronal subpopulations in both white and grey matter. After clearing, the slices are imaged via LSFM with micrometer resolution and in multiple channels simultaneously for a rapid 3D reconstruction. By combining SHORT with LSFM analysis within a routinely high-throughput protocol, it is possible to obtain the 3D cytoarchitecture reconstruction of large volumetric areas at high resolution in a short time, thus enabling comprehensive structural characterization of the human brain.

Author Spotlight: Advancing 3D Cytoarchitecture Analysis - Rapid Volumetric Reconstruction of the Human Brain 

The present protocol provides a step-by-step procedure for rapid and simultaneous optical clearing, muti-round labeling, and 3D volumetric reconstruction of tens of postmortem human brain sections by combining the (SWITCH - H2O2 - Antigen Retrieval - 2,2'-thiodiethanol [TDE]) SHORT tissue transformation technique with light-sheet fluorescence microscopy imaging in a routinely high-throughput protocol.

Despite the numerous clearing techniques that emerged in the last decade, processing postmortem human brains remains a challenging task due to its ...

Neuroscience

High-throughput and Deep-proteome Profiling by 16-plex Tandem Mass Tag Labeling Coupled with Two-dimensional Chromatography and Mass Spectrometry

Isobaric tandem mass tag (TMT) labeling is widely used in proteomics because of its high multiplexing capacity and deep proteome coverage. Recently, an expanded 16-plex TMT method has been introduced, which further increases the throughput of proteomic studies. In this manuscript, we present an optimized protocol for 16-plex TMT-based deep-proteome profiling, including protein sample preparation, enzymatic digestion, TMT labeling reaction, two-dimensional reverse-phase liquid chromatography (LC/LC) fractionation, tandem mass spectrometry (MS/MS), and computational data processing. The crucial quality control steps and improvements in the process specific for the 16-plex TMT analysis are highlighted. This multiplexed process offers a powerful tool for profiling a variety of complex samples such as cells, tissues, and clinical specimens. More than 10,000 proteins and posttranslational modifications such as phosphorylation, methylation, acetylation, and ubiquitination in highly complex biological samples from up to 16 different samples can be quantified in a single experiment, providing a potent tool for basic and clinical research.

Presented here is an optimized high-throughput protocol developed with 16-plex tandem mass tag reagents, enabling quantitative proteome profiling of biological samples. Extensive basic pH fractionation and high-resolution LC-MS/MS mitigate ratio compression and provide deep proteome coverage.

Isobaric tandem mass tag (TMT) labeling is widely used in proteomics because of its high multiplexing capacity and deep proteome coverage. Recently, an ...

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 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 ...

Preparation of Human Tissues Embedded in Optimal Cutting Temperature Compound for Mass Spectrometry Analysis

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