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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Cancer Research

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

Published: April 27th, 2021

DOI:

10.3791/62552

1Department of Biology, Virginia Commonwealth University, 2VCU Lipidomics/Metabolomics Shared Resource, Virginia Commonwealth University School of Medicine, 3Massey Cancer Center

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 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.

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,.css-f1q1l5{display:-webkit-box;display:-webkit-flex;display:-ms-flexbox;display:flex;-webkit-align-items:flex-end;-webkit-box-align:flex-end;-ms-flex-align:flex-end;align-items:flex-end;background-image:linear-gradient(180deg, rgba(255, 255, 255, 0) 0%, rgba(255, 255, 255, 0.8) 40%, rgba(255, 255, 255, 1) 100%);width:100%;height:100%;position:absolute;bottom:0px;left:0px;font-size:var(--chakra-fontSizes-lg);color:#676B82;}

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De-identified human lung tissues were obtained from the Virginia Commonwealth University (VCU) Tissue and Data Acquisition and Analysis Core under an internal review board (IRB) approved protocol (#HM2471). The use of mice for research and harvesting of mice tissues was approved by the VCU institutional animal care and use committee (IACUC).

1. Preparation of materials

NOTE: These steps should be performed a day prior to the tissue washing.

  1. Pre-label and.......

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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.......

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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.......

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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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Name Company Catalog Number Comments
1 mL polypropylene pipette tips NA NA Used to retrieve specimens
1.5 mL polypropylene centrifuge tubes NA NA
10 mL Erlenmeyer flask VWR 89091-116 Used for tube and tissue weighing
AB Sciex Analyst 1.6.2 Sciex NA Software to analyze and integrate MS data
Ammonium formate Fisher Scientific A11550 For LC mobile phases
Analytical scale NA NA Scale that is accurate to 0.1 mg
Bottle top dispenser Sartorius LH-723071 Used for dispensing solvents
C12-Ceramide (d18:1/C12:0); N-(dodecanoyl)-sphing-4-enine Avanti Polar Lipids LM2212 Internal standard
C12-glucosylceramide (d18:1/12:0); N-(dodecanoyl)-1-β-glucosyl-sphing-4-eine Avanti Polar Lipids LM2511 Internal standard
C12-lactosylceramide (d18:1/12:0); N-(dodecanoyl)-1-ß-lactosyl-sphing-4-ene Avanti Polar Lipids LM2512 Internal standard
C12-Sphingomyelin  (d18:1/C12:0), N-(dodecanoyl)-sphing-4-enine-1-phosphocholine Avanti Polar Lipids LM2312 Internal standard
CHLOROFORM OMNISOLV 4L VWR EM-CX1054-1
ClickSeal Biocontainment Lids Thermo Scientific 75007309 To prevent biohazard aeresols during centrifugation
Conflikt Decon Labs 4101 Decontaminant
CTO-20A/20AC Column Oven Shimadzu NA For LC
d17:1-Sphingosine;  (2S,3R,4E)-2-aminoheptadec-4-ene-1,3-diol Avanti Polar Lipids LM2000 Internal standard
d17:1-Sphingosine-1-phosphate; heptadecasphing-4-enine-1-phosphate Avanti Polar Lipids LM2144 Internal standard
DGU20A5R degasser Shimadzu NA
Disposable Culture Tubes 13x100mm VWR 53283-800 13x100 mm screw top tubes
Heated water bath NA NA For overnight lipid extraction
Homogenizer 150 Fisher Scientific 15-340-167 triturate tissues
Homogenizer Plastic Disposable Generator Probe Fisher Scientific 15-340-177 for homogenization
Kimwipes Kimtech 34120 Laboratory grade tissue used to make wicks
Methanol LC-MS Grade 4L VWR EM-MX0486-1
Nexera LC-30 AD binary pump system Shimadzu NA For LC-MS
Permanent marker VWR 52877-310
Phenolic Screw Thread Closure, Kimble Chase (caps for disposable culture tubes) VWR 89001-502 13x100 mm screw top tube caps
Phosphate bufffered saline Thermo Scientific 10010023 To retrieve specimens from tubes after washing
Repeater pipette Eppendorf 4987000118 To dispense LC-MS internal standards
Screw Caps, Blue, Red PTFE/White Silicone VWR 89239-020 Autoinjector vial caps
Screw Thread Glass Vials with ID Patch VWR 46610-724 Autoinjector vials
SIL-30AC autoinjector Shimadzu NA
SpeedVac Thermo Scientific SPD2030P1220 For drying solvents
Supelco 2.1 (i.d.) x 50 mm Ascentis Express C18 column Sigma Aldrich 53822-U For LC-MS
Triple Quad 5500+ LC-MS/MS System Sciex NA For LC-ESI-MS/MS
Ultrasonic water bath Branson Model 2800 for homogenization and resuspension of extracted and dried lipids
Vortexer NA NA For sOCTrP and resuspending dried lipids
VWR Culture Tubes Disposable Borosilicate Glass VWR 47729572 13x100 glass culture tubes
Water Hipersolve Chromanorm LC-MS VWR BDH83645.400

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