Metabolic profiling workflow for cell extracts by targeted hydrophilic interaction liquid chromatography-tandem mass spectrometry

In this study, a targeted approach with wide metabolite coverage was developed for cellular metabolomic analysis using a UHPLC-QTrap-MS system operated in the scheduled multiple reaction monitoring (sMRM) mode. MRM ion pairs were acquired from HeLa cell samples through untargeted analysis using UHPLC-QTOF-MS with SWATH acquisition complemented by missing metabolites from pathway databases. Four different cell extraction protocols were studied and compared based on an experiment series involv-ing the calculation of individual metabolite recoveries (pre/post extraction spiking U -13 C isotope-labeled standards), with a Methanol/Water extraction mixture (1:1; v/v) showing the best results. Two HILIC-MS methods employing a Waters Premier BEH Amide column were developed, utilizing two different chromatographic conditions (20 mM ammonium formate as buffer additive adjusted to a pH = 3.5 with formic acid in ESI+ mode and 20 mM ammonium acetate adjusted to a pH = 7.5 with acetic acid in ESI- mode. One hundred sixty-one (161) metabolites were successfully detected in ESI + mode, whereas 92 were detected in negative ionization mode, totaling to a number of 253 compounds in three differ-ent biological matrices covered by the analytical system employed. Both established HILIC methods were calibrated and validated based on 105 authentic chemical standards and U -13 C-labeled Pichia pastoris (Ko-magataella phaffii) yeast extract as internal standards for cellular matrix (HeLa cells). Within-day and between-day precision was determined on three different QC concentration levels and was below 15% for the entirety of the analytes. Inter-and intra-day accuracies showed values in the range between 85 and 115% (assessed as % recovery) in the entire range. Matrix effects, extraction recoveries and process effi-ciencies were evaluated following the Matuszewski protocol with U -13 C-labeled Pichia pastoris metabolite extract as internal standards. Eventually, the method was utilized to quantify metabolites in HeLa cell extracts.(c) 2022 Elsevier B.V. All rights reserved.

Automated summary

In this study, a targeted approach with wide metabolite coverage was developed for cellular metabolomic analysis using a UHPLC-QTrap-MS system operated in the scheduled multiple reaction monitoring (sMRM) mode. The study compared four different cell extraction protocols and developed two HILIC-MS methods. A total of 161 metabolites were successfully detected in ESI+ mode, and 92 were detected in negative ionization mode. The developed methods were calibrated and validated, showing good accuracy and precision.