Abstract
A novel approach to the analysis of mass spectrally assayed stable isotope-labeling experiments for studies of biosynthetic pathways is reported. This method determines in a mixture of product molecules, the relative number of product molecules synthesized from the stable labeled precursor pathway and those that were either present prior to the labeling period or were produced by an alternate pathway during the course of an experiment. In addition, the isotopic enrichment of the labeled atoms in the product molecules produced from the stable labeled precursor is determined. These isotopic enrichments represent the isotopic enrichment in the immediate precursors which form the product molecules and would reflect any cellular compartmentation of precursor pools. The feasibility of the method using 15NH4Cl and L-[5-15N]glutamine as precursors to study the de novo pyrimidine biosynthetic pathway in isolated rat hepatocytes is demonstrated. The results of these studies show that after incubation of rat hepatocytes with either precursor it is possible to determine the fraction of the uracil nucleotide pool that is formed by the de novo pathway during the period of exposure. The pattern of 15N labeling in the N1 and N3 positions in the uracil moiety is different for the two precursors; however, in most cases the 15N enrichment of each position remained relatively constant for each precursor with either time (15-120 min) or precursor concentration (1 to 10 mM). This method will allow the actual quantitation and isotopic enrichment of product formed by a specific biosynthetic pathway during the course of an experiment and, as such is an improvement over existing labeling techniques.
Highlights
A novel approach to the analysisof mass spectrally zymes and small molecules necessitates experimentation with assayed stable isotope-labeling experiments for studies intact cells and, at the same time, complicates the interpreof biosynthetic pathways is reported
The average spectral analysis of the uracil contained in theuracil nucleoof these two values,43.3 atom % 15N,compares favorably with tide pool of isolated rat hepatocytewshich had been incubated the overall 44.8 atom % 15N reported by Merck, Sharp and with either 15NH4C1or ~-[5-"N]glutamine should yield the Dohme
Althoughconsiderable variation was observed in 15N enrichment of the N3 positionfor the various time points after ~-[5-"N]glutamine incubatio(nFig. 6C),the patternwas again similar to that observed for the concentration experiments (Fig. 5C). 30-120 min after incubation of hepatocytes with 1mM l4NH4Cl the fractionof uracil produced from the precursor (Fig. 6B)appeared to reach a maximum and remain relatively constant after[30] min
Summary
We have chosenI5Nas the labeled atoms, the model and derivation of equations is similar for any combinationof 2 atoms in themolecule. In order to obtain three independent equations which can be solved simultaneously for F, N1, and N3 it is necessary to determine the relative ion abundances for twoion clusters produced by electron impact mass spectromet~ from C,H,Y,O, and mixtures of C,N,N,O, and C,H,NCy.2)0,N1N3 type molecules, respectively.The ion clusters used for isotopic abundance measurements follow. Can be divided into those formed (F) from the labeled precur- Each of the ion currents mii has a probabilityof occurrence' sor pools A*B/AB and A*C/AC and thefraction (1-F) which shown schematicallyin Fig. 3A for the ion clusters with both includesmoIecules ABCA containing only natural isotopic N l and N3, abundances. The method should be applicable for any product molecule formedfromat least two isotopically enriched precursor molecules.,in this report the derivation and applications of equations will be restricted to molecules of the ABCA type. Values for+l/40,&/&,and yl/yo were obtained from reference uracil or uracil extracted from biological extracts which were not exposed to stable labeledprecursors. ao/yo and K ~ / +we~re determined from measurements of both ref-
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