A simple method for sphingolipid analysis of tissues embedded in optimal cutting temperature compound

Abstract

MS-assisted lipidomic tissue analysis is a valuable tool to assess sphingolipid metabolism dysfunction in disease. These analyses can reveal potential pharmacological targets or direct mechanistic studies to better understand the molecular underpinnings and influence of sphingolipid metabolism alterations on disease etiology. But procuring sufficient human tissues for adequately powered studies can be challenging. Therefore, biorepositories, which hold large collections of cryopreserved human tissues, are an ideal retrospective source of specimens. However, this resource has been vastly underutilized by lipid biologists, as the components of OCT compound used in cryopreservation are incompatible with MS analyses. Here, we report results indicating that OCT compound also interferes with protein quantification assays, and that the presence of OCT compound impacts the quantification of extracted sphingolipids by LC-ESI-MS/MS. We developed and validated a simple and inexpensive method that removes OCT compound from OCT compound-embedded tissues. Our results indicate that removal of OCT compound from cryopreserved tissues does not significantly affect the accuracy of sphingolipid measurements with LC-ESI-MS/MS. We used the validated method to analyze sphingolipid alterations in tumors compared with normal adjacent uninvolved lung tissues from individuals with lung cancer and to determine the long-term stability of sphingolipids in OCT compound-cryopreserved normal lung tissues. We show that lung cancer tumors have significantly altered sphingolipid profiles and that sphingolipids are stable for up to 16 years in OCT compound-cryopreserved normal lung tissues. This validated sphingolipidomic OCT compound-removal protocol should be a valuable addition to the lipid biologist’s toolbox.

Comparative liquid chromatography/tandem mass spectrometry lipidomics analysis of macaque heart tissue flash‐frozen or embedded in optimal cutting temperature polymer (OCT): Practical considerations

“…Previously, Rohrbach et al 11 validated a procedure to analyze sphingolipid alterations in OCT‐embedded human tissues. Our findings agree with Rohrbach's results, but, importantly, it offers greater coverage of lipid classes.…”

“…The mass spectral data was normalized by tissue weight after OCT removal 11 . The DDA data were processed with Progenesis QI™ software (NonLinear Dynamics, UK).…”

Lipidomic Profiling Reveals Biological Differences between Tumors of Self-Identified African Americans and Non-Hispanic Whites with Cancer

“…In cancers, it is well established that dysregulation of sphingolipid metabolism and of their signaling pathways contributes to disease development, progression, and responses to therapy. Tumors of human patients with various types of cancer have significant alterations of sphingolipid levels as compared to normal adjacent tissues [ 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 ]. There is also broad consensus that key sphingolipid-metabolism-regulating enzymes impact malignancy, responses to therapy, and prognosis, and regulators of sphingolipid levels are considered important therapeutic targets in cancer, with several being tested in clinical trials [ 8 , 21 , 22 , 23 ].…”

“…In addition, given the recognized importance of sphingolipid alterations in the etiology of cancer and their therapeutic potential, precisely establishing how sphingolipid metabolism is altered may lead to the development of targeted therapies. Therefore, to decrease the gap in our understanding of the reprogramming of lipid metabolism in the tumors of individuals self-identifying as AA, we used methodology we previously developed [ 15 , 16 ] to conduct a modern sphingolipidomic analysis of the normal adjacent uninvolved (abbreviated “unin.” henceforth) tissues and tumors of self-identified AA and NHW males with lung adenocarcinoma (LUAD), colorectal adenocarcinoma (COAD), liver adenocarcinoma (HCC), and head and neck squamous cell carcinoma (HNSCC) and of AA and NHW females with endometroid endometrial carcinoma (EEC).…”

Removal of optimal cutting temperature (O.C.T.) compound from embedded tissue for MALDI imaging of lipids

“…and whose access to alternatives is limited. To address this, some [30,[36][37][38][39] have attempted to remove O.C.T. from embedded samples to improve compatibility with MS and restore a maximum analyte ion yield.…”