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.
Highlights
MS-assisted lipidomic tissue analysis is a valuable tool to assess sphingolipid metabolism dysfunction in disease
In sphingolipidomic OCT compoundremoval protocol (sOCTrP), tissues are washed several times prior to MS analysis, and we have suggested that tissues can be “wet-weighed” as a method to externally normalize data
Much is to be learned of the roles that sphingolipids play in human cancers, and large-scale studies that characterize sphingolipid metabolism alterations in tissues can greatly enhance our ability to probe the mechanistic underpinnings of these diseases
Summary
MS-assisted lipidomic tissue analysis is a valuable tool to assess sphingolipid metabolism dysfunction in disease. 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. Sphingolipid dysregulation is known to significantly contribute to cancer initiation, progression, response to therapy, and the development of multi-drug resistance [3, 10]. Given this broad impact on the etiology of disease, there is great interest in precisely establishing disease-specific sphingolipid alterations.
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