Double Cyclization Tandem Mass for Identification and Quantification of Phosphatidylcholines Using Isobaric Six-Plex Capillary nLC-MS/MS

J Am Soc Mass Spectrom. 2024 Jul 3;35(7):1403-1412. doi: 10.1021/jasms.3c00447. Epub 2024 Jun 13.

Abstract

Multiplexing of phosphatidylcholine analysis is hindered by a lack of appropriate derivatization. Presented here is a tagging scheme that uses a quaternary amine tag and targets the hydroxy group of the phosphate, which switches the net charge from neutral to +2. Quantitative yields were achieved from >99% reaction completion derived by dimethoxymethyl morpholinium (DMTMM) activation. Fragmentation of phosphatidylcholines (PCs) and lysophosphatidylcholines (LPCs) releases two trimethylamines and the acyl chains through neutral loss and generates a unique double cyclization constant mass reporter. Selective incorporation of isotopes onto the tag produces a six-plex set of isobaric reagents. For equivalent six-plex-labeled samples, <14% RSD was achieved, followed by a dynamic range of 1:10 without signal compression. Quantification of PCs/LPCs in human hepatic cancer cells was conducted as six-plex using data-dependent analysis tandem MS. We report a six-plex qualitative and quantitative isobaric tagging strategy expanding the limits of analyzing PCs/LPCs.

MeSH terms

  • Cell Line, Tumor
  • Chromatography, Liquid / methods
  • Cyclization
  • Hep G2 Cells
  • Humans
  • Lysophosphatidylcholines / analysis
  • Lysophosphatidylcholines / chemistry
  • Phosphatidylcholines* / analysis
  • Phosphatidylcholines* / chemistry
  • Tandem Mass Spectrometry* / methods

Substances

  • Phosphatidylcholines
  • Lysophosphatidylcholines