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Volume 72, Issue 6

High-throughput quantification of microbial-derived organic acids in mucin-rich samples via reverse phase high performance liquid chromatography Open Access

Abstract

Organic acids (short chain fatty acids, amino acids, etc.) are common metabolic byproducts of commensal bacteria of the gut and oral cavity in addition to microbiota associated with chronic infections of the airways, skin, and soft tissues. A ubiquitous characteristic of these body sites in which mucus-rich secretions often accumulate in excess, is the presence of mucins; high molecular weight (HMW), glycosylated proteins that decorate the surfaces of non-keratinized epithelia. Owing to their size, mucins complicate quantification of microbial-derived metabolites as these large glycoproteins preclude use of 1D and 2D gel approaches and can obstruct analytical chromatography columns. Standard approaches for quantification of organic acids in mucin-rich samples typically rely on laborious extractions or outsourcing to laboratories specializing in targeted metabolomics. Here we report a high-throughput sample preparation process that reduces mucin abundance and an accompanying isocratic reverse phase high performance liquid chromatography (HPLC) method that enables quantification of microbial-derived organic acids. This approach allows for accurate quantification of compounds of interest (0.01 mM – 100 mM) with minimal sample preparation, a moderate HPLC method run time, and preservation of both guard and analytical column integrity. This approach paves the way for further analyses of microbial-derived metabolites in complex clinical samples.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): amino acids , glycoproteins , high performance liquid chromatography , mucin , organic acids , reverse phase liquid chromatography and targeted metabolomics
Funding
This study was supported by the:
  • National Institute of Dental and Craniofacial Research (Award T90DE0227232)
    • Principle Award Recipient: SarahK Lucas
  • National Heart, Lung, and Blood Institute (Award 2T32HL007741-21)
    • Principle Award Recipient: AlexR Villareal
  • National Heart, Lung, and Blood Institute (Award HL136919-03S1)
    • Principle Award Recipient: AlexR Villareal
  • National Heart, Lung, and Blood Institute (Award 1R01HL136919)
    • Principle Award Recipient: RyanC Hunter
© 2023 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.
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2023-06-09
2024-05-05
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High-throughput quantification of microbial-derived organic acids in mucin-rich samples via reverse phase high performance liquid chromatography
J. Med. Microbiol. 72, 001708 (2023); https://doi.org/10.1099/jmm.0.001708
/content/journal/jmm/10.1099/jmm.0.001708
/content/journal/jmm/10.1099/jmm.0.001708
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