Abstract Experimental Description Single-Point

A targeted metabolomic workflow for the determination of positional isotopomer distributions of labeled metabolites in stable isotope tracer studies Thomas D. Horvath1, Shai Dagan2, Di Du1, David H. Hawke1, John N. Weinstein1, Philip L. Lorenzi1, and Patricia Y. Scaraffia3 The Proteomics and Metabolomics Core Facility at The University of Texas MD Anderson Cancer Center, Houston, TX 77054; 2 Israel Institute for Biological Research (IIBR), Ness Ziona, 74100, Israel; 3 Department of Tropical Medicine, Tulane University, New Orleans, LA 70112

1

Stable isotope-labeled substrate compounds (e.g., [13C6]glucose, [13C5,15N2]glutamine, [13C3]lactate) are used as metabolic probes to study the interconnectedness, alteration, or dysfunction of metabolic pathways in biological systems, specific diseases, or under a specific set of experimental conditions such as hypoxia, nutrient status, and drug treatment. Through the careful characterization of collision-induced dissociation (CID) molecular ion fragments, and judicious selection of selected reaction monitoring (SRM) transitions, modern liquid chromatography/tandem mass spectrometry (LC/MS-MS) instrumentation is capable of measuring the positional isotopomer distribution (e.g., [1-13C1]Alanine (Ala), [2-13C1]Ala, [3-13C1]Ala) of isotope labeled metabolites within a given isotopolog distribution (e.g., [13C0]Ala to [13C3]Ala) for any targeted metabolite pool (Ma et al., 2013). Here we demonstrate a targeted metabolomic workflow that was used to determine the positional isotopomer distributions of [13C]atom incorporation into Ala, proline (Pro), glutamine (Gln), and glutamate (Glu) produced in Aedes aegypti mosquitoes fed [1,2-13C2]glucose over a 24 h time course; only the experimental parameters and data for Ala are presented here.

Quantification for the total level (naturally abundant, isotopically labeled, background Ala isotopologs plus enriched [13C]Ala isotoplogs) of each unlabeled and each isotope-labeled positional isotopomer for each isotopolog of Ala was performed using a single-point calibration method that used the following equation:

Experimental Description Mosquito rearing and maintainance, feeding, and sample collection and processing methods are described in detail in a recently accepted manuscript (Horvath et al., 2018). Sample analysis was performed using an Agilent LC/MS-MS comprised of an Infinity 1290 LC coupled to a 6460 tandem mass spectrometer operated in the dynamic multiple reaction monitoring (dMRM) scan mode. For example, a total of 9 SRM transitions (Table 1) were monitored to account for all of the possible permutations of [13C]atom incorporation into Ala for the mosquito study samples that were fed [1,2-13C2]glucose. Three experimental parameters were determined for each metabolite monitored in order to make mathematical corrections to the empirical data: i.) total metabolite content in the sample (e.g., [Ala]Total = [[M+0]Ala] + [[M+1]Ala] + [[M+2]Ala] + [[M+3]Ala]); ii.) natural abundance proportion (PNA) of each successive level of isotope labeling (e.g., PNA,([M+1]Ala) = [[M+1]Ala]/[Ala]Total); and iii.) fragmentation-specific positional probability (PPOS) factors for each individual [13C]Ala SRM transition to properly account for the distribution of specific carbon positions between the product ion and various neutral loss fragments. The naturally occurring background ensemble of [13C], [15N], [18O], or [2H]atom labeled metabolites in the biological sample was subtracted from the empirically determined concentrations using a custom algorithm derived by our laboratory. Ultimately, positional isotopomer distribution data for mosquitoes fed [1,2-13C2]glucose were indicative of metabolic flux through multiple specific pathways.

Eq. 1 where: ● [[M+x]Ala] is the measured concentration of a particular Ala positional isotopomer, that is designated by a particular SRM transition (e.g., m/z 91 -> m/z 45 for [M+1]Ala), for a given [M+x]Ala isotopolog distribution (e.g., including [13C]background, [2H], [15N], or [18O], and [13C]enriched labeled Ala). ● x is the isotope labeling state (number of atoms labeled) for each Ala isotopolog (e.g. 0, unlabeled Ala; +3, Ala with three isotope-labeled atoms). ● The [[13C0]AlaCalStd], [[13C3,15N1]AlaIS], and DF are defined as the nominal concentration of the calibration standard (5 µM), the IS (2.5 µM), and the dilution factor, respectively. ● The mean instrument response factor (IRF) is defined as the ratio of the integrated Ala peak area for a specific MS-MS transition over the integrated IS peak area for a specific Ala-IS MS-MS transition. To determine the level of enrichment of the positional isotopomers for [ C]Ala over the background ensemble of [ C]Ala, [ H]Ala, [15N]Ala, or [18O]Ala, the following equation was used: 13

where: ● [[M+x]Ala] is the measured concentration of a particular Ala positional isotopomer, see Eq. 1 ● x is the isotope labeling state, see Eq. 1 ● PPOS is the fragment specific positional probability factor that accounts for the distribution of different carbon positions between the product ion and the neutral loss fragments, see table 1 for these empirically determined values. ● PNA,([M+1]Ala is the natural abundance proportion for each successive level of isotope labeling, see Table 1 for these values.

Application of the Correction Method to Empirical Data

Precursor ion (m/z)a

Product ion (m/z)

Product ion formula

CEb (v)

[13C0]Ala

90

44

2,3-C2H6N1

6

[ C0]Ala

90

29

2-C1H3N1

50

[ C1]Ala

91

44

2,3-C2H6N1

6

*

[ C1]Ala

91

45

2,3-C2H6N1

6

[13C2]Ala

92

45

2,3-C2H6N1

6

0.666

[13C2]Ala*

92

46

2,3-C2H6N1

6

0.333

[13C2]Ala

92

29

2-C1H3N1

50

[13C2]Ala*

92

30

2-C1H3N1

50

[13C3]Ala

93

46

2,3-C2H6N1

6

13

13

13

P

d NA

0.963 0.0356

0.00124

PPOS

e

-

0.333 0.666

0.333 0.666

0.00015**

1

[13C3,15N1]Ala 2,3-C2H6N1 94 47 6 c (IS) *Isotopolog product ion contains an additional [13C]atom. **The instrument response for [13C3]Ala was below the detection limit for most samples, so a theoretical value was calculated using the following website: www.sisweb.com/mstools/isotope.htm. Abbreviations: amass-to-charge ratio; bCollision Energy; cInternal Standard; dnatural abundance proportion; epositional probability factor

Concentration of Ala isotopologs in the mosquito whole body sample extracts (µM)

Ala Isotopologs and IS

13

Eq. 2

150

Table 1. Precursor ions, product ions, product ion formula, and collision energies used for monitoring the [13C]isotopologs and internal standard (IS) for Ala. The empirically determined natural abundance proportion (PNA) and fragment specific positional probability (PPOS) factors that were used to make the Ala isotopolog corrections were calculated from the bovine blood meal (BM) and BM + 100 mM glucose mosquito control samples.

Corrected Time Course Data

Ala

Legend

O H 3C

3

100

2

1

[[M+1]Ala] [[13C1]Ala] [[M+2]Ala], HM [[13C2]Ala], HM [[M+2]Ala], LM [[13C2]Ala], LM [[M+3]Ala

OH

NH2

[[13C3]Ala]

50

0 0

6

12 Time (h)

18

24

Figure 1. Plot of the concentration of Ala isotopologs (µM) contained in mosquitoes fed [1,2-13C2]glucose as a function of Time (h) from one of the three replicate studies. The empirical Ala isotopolog data measured in the mosquito extracts are represented by the dashed lines and open symbols. The corrected Ala isotopolog data (corrected using Eq. 2) are represented by the solid lines and shaded symbols. The inset contains the chemical structure of Ala and includes the numbers for the carbon atoms on the molecule. In the Legend, the abbreviations HM and LM correspond to the High-Mass fragments (contains carbons C2 and C3; 2,3-C2H6N1) and Low-Mass fragment (contains carbon C2 only; 2-C1H3N1), respectively.

2

A.

[[13C1]Ala] (nmol/mosquito)

Single-Point Calibration and Correction Methods

[[13C1]Ala] (m/z: 91 - > 45) H313C

2.0

or

CH H 3N

1.5

O

13CH

H 3N

OH

OH

m/z 45

m/z 45

[[13C1]Ala] (m/z: 91 - > 44)

1.0

O

H 3C CH

0.5 H 3N

13C

OH

m/z 44

0

B.

H 3C

O

0

[[13C2]Ala] (nmol/mosquito)

Abstract

Legend for Fig.2 Panel A

6

12

Time (h)

18

24

Legend for Fig.2 Panel B [[13C2]Ala] (LM; m/z: 92.1 - > 30.0)

[[13C2]Ala] (HM; m/z: 92.1 - > 46.0)

10

H313C

H313C

O

13CH

13CH

OH

H 3N

CH H 3N

m/z 45

or

13C

O

H 3C

O

H313C

C

H 3N

OH

13

CH

H 3N

OH

m/z 45

OH

m/z 30

m/z 30

[[13C2]Ala] (HM; m/z: 92.1 - > 45.0)

O

13CH 13C

H 3N

m/z 46

5

H 3C

O

[[13C2]Ala] (LM; m/z: 92.1 - > 29.0) H313C

13C

O CH

OH

m/z 29

H 3N

13C

OH

0 0

6

12

18

24

Time (h) Figure 2. Panel A: Plot of positional isotopomer concentrations (nmol/mosquito) of [13C1]Ala as a function of Time (h) in mosquitos fed [1,2-13C2]glucose. As illustrated, the [13C]atom (designated as red text on the structural diagrams in the Legend) can be located at carbon positions C2 or C3, and be included in the product ion fragment ( ), or, the [13C]atom can be located at C1, and be lost in the neutral loss fragment ( ); Panel B: Plot of positional isotopomer concentrations (nmol/mosquito) of [13C2]Ala as a function of Time (h) in mosquitos fed [1,2-13C2]glucose. The two [13C]atoms can be distributed among the three possible carbon positions for Ala in the following two High-Mass (HM) configurations that include carbon positions C2 and C3 (2,3-C2H6N1) in the product ion fragment: i) both [13C]atoms are located at C2 or C3, and are included in the product ion fragment ( ); ii) one [13C]atom is located in C2 or C3, and is included in the product ion fragment, and one [13C]atom is located at C1 and is lost in the neutral loss fragment ( ). Additionally, the following two Low-Mass (LM) configurations include only position C2 (2-C1H3N1) in the product ion fragment: i) one [13C]atom is located at C2, and is included in the product ion fragment ( ); ii) both [13C]atoms are located at positions C1 and C3, and both are lost in the neutral loss fragments ( ). The red lines in the Legends indicate the carbon-carbon bonds that are broken during the fragmentation process that takes place within the collision cell of the mass spectrometer.

Conclusions

In the model biological system studied, the resulting positional isotopomer distribution data provide information regarding the complex network of biochemical pathways that may yield common metabolic products to the metabolite pool under the specific set of experimental conditions (Horvath et al, 2018). This approach is useful for distinguishing different kinetic routes for incorporation of [13C]atoms at specific positions in targeted metabolites.

Acknowledgments

We thank Mrs. Lin Tan for technical assistance. This work was financially supported by the Corine Adams Baines Professorship Award, U.S. National Institutes of Health, National Institute of Allergy and Infectious Diseases Grant R01AI088092 (to PYS), NIH 1S10OD012304-01, Cancer Prevention Research Institute of Texas (CPRIT) Grant RP130397, and The University of Texas MD Anderson’s NCI Cancer Center Support Grant CA016672.

References

1. Ma, X., Dagan, S., Somogyi, A., Wysocki, V. H., and Scaraffia, P. Y. (2013) Low mass MS/MS fragments of protonated amino acids used for distinction of their 13C-isotopomers in metabolic studies. J Am Soc Mass Spectrom 24, 622-631 2. Horvath, T.D., Dagan, S., Lorenzi, P.L., Hawke, D.H., Scaraffia, P.Y. (2018) Positional stable isotope tracer analysis reveals carbon routes during ammonia metabolism of Aedes aegypti mosquitoes. FASEB J. (In Press)