Comparison of ESI‐MS/MS and APCI‐MS methods for the quantification of folic acid analogs in C. elegans

Abstract

Folic acid (FA) plays a vital role in central metabolism, including the one carbon cycle, nucleotide, and amino acid biosynthesis. The development of sensitive, accurate analytical methods to measure FA intermediates in tissues is critical to understand their biological roles in diverse physiological and pathological contexts. Here, we developed a highly sensitive method for the simultaneous quantification of FA intermediates in the nematode Caenorhabditis elegans as a model to dissect metabolic networks. The method was further validated by analyzing the worm folate pool upon RNAi knockdown of the dihydrofolate reductase gene dhfr‐1. Comparative mass spectrometry behavior of the FA analogs using two different ion sources, electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI), revealed ESI‐MS/MS to be more sensitive, but APCI‐MS provided more detailed structure inferences, which can elucidate chemical investigation and synthesis of FA analogs. Finally, we report on the use of in vitro oxidation coupled with high‐resolution mass spectrometry as a tool to discover new endogenous FA derivatives in the nematode.

Regulation of the one carbon folate cycle as a shared metabolic signature of longevity

“…We then measured the levels of folate cycle intermediates upon dhfr-1i knockdown. dhfr-1 RNAi led to an accumulation of upstream FA, concomitant with a reduction in downstream intermediates THF, 5MTHF, MN, ML, and FO, confirming our previous results 18 . Consistent with a critical role as rate-limiting enzyme in the one-carbon cycle, supplementation of dhfr-1i treated worms with the downstream intermediate 5MTHF restored near normal levels of all folate cycle intermediates, except for upstream FA which remained high.…”

“…Synchronized young adults for each strain were collected (approx. three plates) in five biological replicates in single tubes and washed three times with buffer solution M9 and 0.1% of butyl hydroxy toluene (BHT) was added to prevent auto oxidation as previously reported on our work 18,62 . Samples were snap frozen in liquid nitrogen and stored at −80°C before use.…”

“…Identification and relative quantification of folic acid intermediates were performed on a triple quadrupole mass spectrometer (QqQMS) (TSQ Altis, ThermoFisher Scientific GmbH, Bremen, Germany), as previously published by our group 18 . Data was analyzed using Xcalibur version 4.0.…”

Optimization of mass spectrometry settings for steroidomic analysis in young and old killifish

“…In both methods, samples were afterwards rotated for 1 h at 4°C and centrifuged for 10 min at 10.000 rpm. The supernatant was transferred to a new tube in both methods and dried using a speedvac as described in our previous works [34,35]. The third method used SPE to enrich the sterol fraction: H L B p r i m e c a r t r i d g e s ( O a s i s , Wa t e r s ) w e r e preconditioned with 1 mL methanol and 1 mL water with 1% v/v formic acid before adding the sample.…”

High-dose folic acid supplementation results in significant accumulation of unmetabolized homocysteine, leading to severe oxidative stress in Caenorhabditis elegans

“…In fact, C. elegans has a series of orthologous genes that encode enzymes involved in the mammalian folate and methionine cycles, as shown in Fig. 1 [ [15] , [16] , [17] , [18] , [19] , [20] , [21] , [22] ]. Thus, worms would be used as a model animal to reach an improved understanding of the relationship between biological events and supplementation with folic acid.…”