Comprehensive Isotopic Targeted Mass Spectrometry: Reliable Metabolic Flux Analysis with Broad Coverage

Metabolic flux analysis
(MFA) is highly relevant to understanding
metabolic mechanisms of various biological processes. While the pace
of methodology development in MFA has been rapid, a major challenge
the field continues to witness is limited metabolite coverage, often
restricted to a small to moderate number of well-known compounds.
In addition, isotopic peaks from an enriched metabolite tend to have
low abundances, which makes liquid chromatography tandem mass spectrometry
(LC-MS/MS) highly useful in MFA due to its high sensitivity and specificity.
Previously we have built large-scale LC-MS/MS approaches that can
be routinely used for measurement of up to ∼1,900 metabolite/feature
levels [Gu et al. Anal.
Chem. 2015, 87, 12355−12362. Shi et al. Anal. Chem. 2019, 91, 13737–13745.]. In this study, we aim to expand our previous
studies focused on metabolite level measurements to flux analysis
and establish a novel comprehensive isotopic targeted mass spectrometry
(CIT-MS) method for reliable MFA analysis with broad coverage. As
a proof-of-principle, we have applied CIT-MS to compare the steady-state
enrichment of metabolites between Myc­(oncogene)-On and Myc-Off Tet21N
human neuroblastoma cells cultured with U-13C6-glucose medium. CIT-MS is operationalized using multiple reaction
monitoring (MRM) mode and is able to perform MFA of 310 identified
metabolites (142 reliably detected, 46 kinetically profiled) selected
from >35 metabolic pathways of strong biological significance.
Further,
we developed a novel concept of relative flux, which eliminates the
requirement of absolute quantitation in traditional MFA and thus enables
comparative MFA under the pseudosteady state. As a result, CIT-MS
was shown to possess the advantages of broad coverage, easy implementation,
fast throughput, and more importantly, high fidelity and accuracy
in MFA. In principle, CIT-MS can be easily adapted to track the flux
of other labeled tracers (such as 15N-tracers) in any metabolite
detectable by LC-MS/MS and in various biological models (such as mice).
Therefore, CIT-MS has great potential to bring new insights to both
basic and clinical metabolism research.