A Single LC-MS/MS Analysis to Quantify Coa Biosynthetic Intermediates and Short-Chain Acyl Coas

H OH metabolites OH

Article A Single LC-MS/MS Analysis to Quantify CoA Biosynthetic Intermediates and Short-Chain Acyl CoAs

Anthony E. Jones 1 , Nataly J. Arias 1, Aracely Acevedo 1, Srinivasa T. Reddy 1,2, Ajit S. Divakaruni 1,* and David Meriwether 2,3,*

1 Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, 650 Charles E Young Dr. South, Los Angeles, CA 90095, USA; [email protected] (A.E.J.); [email protected] (N.J.A.); [email protected] (A.A.); [email protected] (S.T.R.) 2 Department of Medicine, Division of Cardiology, David Geffen School of Medicine, University of California, Los Angeles, 10833 Le Conte Avenue, Los Angeles, CA 90095, USA 3 Department of Medicine, Division of Digestive Diseases, David Geffen School of Medicine, University of California, Los Angeles, 10833 Le Conte Avenue, Los Angeles, CA 90095, USA * Correspondence: [email protected] (A.S.D.); [email protected] (D.M.)

Abstract: Coenzyme A (CoA) is an essential cofactor for dozens of reactions in intermediary metabolism. Dysregulation of CoA synthesis or acyl CoA metabolism can result in metabolic or neu- rodegenerative disease. Although several methods use liquid chromatography coupled with mass spectrometry/mass spectrometry (LC-MS/MS) to quantify acyl CoA levels in biological samples, few allow for simultaneous measurement of intermediates in the CoA biosynthetic pathway. Here

we describe a simple sample preparation and LC-MS/MS method that can measure both short-chain acyl CoAs and biosynthetic precursors of CoA. The method does not require use of a solid phase

Citation: Jones, A.E.; Arias, N.J.; extraction column during sample preparation and exhibits high sensitivity, precision, and accuracy. Acevedo, A.; Reddy, S.T.; Divakaruni, It reproduces expected changes from known effectors of cellular CoA homeostasis and helps clarify A.S.; Meriwether, D. A Single the mechanism by which excess concentrations of etomoxir reduce intracellular CoA levels. LC-MS/MS Analysis to Quantify CoA Biosynthetic Intermediates and Keywords: LC-MS/MS; CoA biosynthesis; short-chain acyl CoAs; mitochondria; etomoxir Short-Chain Acyl CoAs. Metabolites 2021, 11, 468. https://doi.org/ 10.3390/metabo11080468 1. Introduction Academic Editor: Amedeo Lonardo Coenzyme A (CoA) is an obligatory co-factor in all organisms [1]. It is involved in several aspects of mammalian cellular metabolism including the Krebs cycle, oxidation Received: 18 May 2021 of fatty acids and branched chain amino acids, as well as synthesis of fatty acids and Accepted: 14 July 2021 Published: 21 July 2021 sterols. CoA acts as an acyl group carrier forming thioester linkages with organic acids to yield acyl CoAs (e.g., acetyl CoA or palmitoyl CoA) [1]. The formation of a CoA

Publisher’s Note: MDPI stays neutral thioester serves multiple functions. The large free energy of hydrolysis of the thioester bond with regard to jurisdictional claims in serves as a means to ‘charge’ or ‘activate’ the adjoining acyl group for further metabolism. published maps and institutional affil- Additionally, formation of CoA esters can aid in subcellular metabolite compartmentation. iations. These acyl CoA esters are used as building blocks for biosynthetic reactions, substrates for post-translational modifications, or energy substrates oxidized to ultimately produce ATP [2]. In mammals, de novo synthesis of CoA begins with the cellular uptake of pantothenate (vitamin B ) via the sodium-dependent multivitamin transporter (SMVT) [3,4]. Five Copyright: © 2021 by the authors. 5 Licensee MDPI, Basel, Switzerland. enzymatic reactions convert pantothenate into CoA. Pantothenate is first phosphory- This article is an open access article lated by pantothenate kinase (PANK), the primary rate-controlling step in CoA synthe- distributed under the terms and sis [5]. The subsequent conjugation with cysteine followed by decarboxylation results 0 0 conditions of the Creative Commons in 4 -phosphopantetheine. Lastly, 4 -phosphopantetheine is converted to CoA by the bi- Attribution (CC BY) license (https:// functional enzyme Coenzyme A synthase (COASY). An adenylyl group from ATP is first 0 creativecommons.org/licenses/by/ transferred to 4 -phosphopantetheine to form dephospho-CoA, and followed by phospho- 4.0/). rylation of dephospho-CoA to form unesterified (“free”) CoA [1].

Metabolites 2021, 11, 468. https://doi.org/10.3390/metabo11080468 https://www.mdpi.com/journal/metabolites Metabolites 2021, 11, 468 2 of 18

Free CoA can be used to activate carboxylic acids of differing chain lengths through the action of various CoA ligases [6,7]. Disruption of CoA biosynthesis or short-chain acyl CoA metabolism can result in pathological defects. For example, altering levels of short-chain acyl CoAs disrupts hepatic metabolic homeostasis [8], and certain forms of neurodegeneration are caused by mutations in enzymes responsible for CoA synthesis [9,10]. Short-chain acyl CoAs also have additional roles in cell biology beyond energy metabolism, such as in post-translational modifications. In macrophages, for example, increased acetyl CoA production via ATP citrate lyase (ACLY) is associated with histone acetylation and the epigenetic changes observed during the anti-inflammatory activation with interleukin-4 (IL-4) [11]. Further work has demonstrated that intracellular CoA levels are associated with IL-4-driven macrophage activation [12]. However, the precise mechanism by which this occurs, as well as which short-chain CoA esters are similarly associated with the IL-4 response, remains unclear. A single assay for measuring CoA biosynthetic intermediates as well as short-chain acyl CoA species is therefore important to better understand CoA homeostasis in response to physiologically relevant stimuli such as macrophage activation. Several techniques are available to measure short-chain acyl CoAs. These include fluorescence-based enzymatic assays, high performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), and HPLC coupled with tandem mass spectrometry-based (LC-MS/MS) assays. Of these, LC-MS/MS-based methods offer the highest selectivity and sensitivity [13,14]. Although several published procedures detail the quantification of acyl CoAs of dif- ferent chain lengths [15–22], none currently present a single method for the extraction and analysis of both short-chain acyl CoAs and intermediates in the CoA biosynthetic pathway. A primary challenge for this lies in the preparation of the biological sample. Many LC-MS/MS assays use halogenated carboxylic acids or oxo-acids for deproteinizing the sample (e.g., trichloroacetic acid) [23]. Solid phase extraction (SPE) is then used to purify acyl CoAs and remove the deproteinizing agent before LC-MS/MS analysis [18–20,22]. Mechanistically, SPE operates by retaining the acyl CoA species on the solid phase sorbent while the aqueous phase containing the deproteinization agent is discarded as waste [24]. While SPE efficiently binds relatively hydrophobic acyl CoAs (e.g., propionyl CoA, isovaleryl CoA, etc.), it does not efficiently retain relatively hydrophilic CoA biosynthetic pathway intermediates such as dephospho-CoA and pantothenate. It is therefore difficult to use SPE when measuring both acyl CoAs and CoA biosynthetic intermediates. Their divergent polarities also create difficulty in developing a single LC-MS/MS method. Rela- tively hydrophobic short-chain acyl CoAs exhibit the best chromatography when using traditional C18 columns under reverse phase conditions [6]. Under those same conditions, however, relatively hydrophilic species including pantothenate, dephospho-CoA, and free CoA exhibit poor chromatographic peak shape and retention [17,25]. Here we present a single sample preparation and LC-MS/MS method that allows for the extraction and quantification of both short-chain acyl CoAs as well as the CoA biosynthetic intermediates pantothenate and dephospho-CoA. We use 5-sulfosalicylic acid (SSA) for sample deproteinization, as this compound obviates the need for removal by SPE prior to LC-MS/MS analysis. This therefore retains a significant amount of pantothenate and dephospho-CoA from biological samples that would otherwise be lost following SPE-based purification. We overcome the challenge of poor chromatographic separation between the various species by carefully controlling the pH, thereby minimizing the charge on the competing positively and negatively polarizable moieties of the CoA backbone. We then further suppress and mask remaining polarization and charge with ion-pairing chromatography. When combined with an ultra-high performance liquid chromatography (UHPLC) C18 column with high theoretical plates, we observe stable and symmetric peak shape with good resolution across a range of analytes. We demonstrate that this method is able to reproduce findings generated using other LC-MS/MS methods and also helps to reveal the mechanism by which excess concentrations of the carnitine palmityoltransferase- Metabolites 2021, 11, x FOR PEER REVIEW 3 of 19 Metabolites 2021, 11, 468 3 of 18

is able to reproduce findings generated using other LC-MS/MS methods and also helps to reveal1 (CPT-1) the mechanism inhibitor etomoxirby which excess decreases concentrations intracellular of the CoA carnitine levels palmityoltransfer- in bone marrow-derived ase-1 (CPT-1) inhibitor etomoxir decreases intracellular CoA levels in bone marrow-de- macrophages (BMDMs). rived macrophages (BMDMs). 2. Results 2. Results 2.1. The MRM MS/MS Method Can Detect Both CoA Biosynthetic Intermediates and Short-Chain 2.1.Acyl-CoAs The MRM MS/MS Method Can Detect Both CoA Biosynthetic Intermediates and Short- Chain Acyl-CoAs We initially optimized the mass spectrometry to ensure robust detection of both We initially optimized the mass spectrometry to ensure robust detection of both short-chain acyl CoAs as well as pantothenate and dephospho-CoA (Figure1, Figure S1). short-chain acyl CoAs as well as pantothenate and dephospho-CoA (Figure 1, Figure S1).

FigureFigure 1. 1. TheThe CoA CoA biosynthetic biosynthetic pathway: pathway: the metabolites the metabolites detected by detected the method by thedetailed method in this detailed in manuscriptthis manuscript are highlighted are highlighted in green. Abbreviations: in green. Abbreviations: PANK = pantothenate PANK kinase; = pantothenate PPCS = phospho- kinase; PPCS = pantothenoylcysteine synthase; PPCDC = phosphopantothenoylcysteine decarboxylase; PPAT = phosphopantothenoylcysteine synthase; PPCDC = phosphopantothenoylcysteine decarboxylase; phosphopantetheine adenylyl transferase; and DPCK = dephosphocoenzyme A kinase. The bifunc- tionalPPAT enzyme = phosphopantetheine Coenzyme A synthase adenylyl (COASY) transferase; is comprised and of DPCK PPAT = and dephosphocoenzyme DPCK. A kinase. The bifunctional enzyme Coenzyme A synthase (COASY) is comprised of PPAT and DPCK.

To optimize the MS detection of short-chain acyl CoAs, we prepared 1 µg/mL solutions of each acyl CoA standard in the SSA extraction solution (see Methods) and these standards were directly infused into the mass spectrometer for MS/MS analysis. We focused on optimizing the detection of short-chain acyl CoAs with the MS operated in positive mode, as previous studies demonstrated that they are more efﬁciently ionized under Metabolites 2021, 11, x FOR PEER REVIEW 4 of 19

To optimize the MS detection of short-chain acyl CoAs, we prepared 1 μg/mL solutions of each acyl CoA standard in the SSA extraction solution (see Methods) and these Metabolites 2021, 11, 468 standards were directly infused into the mass spectrometer for MS/MS analysis. We4 of fo- 18 cused on optimizing the detection of short-chain acyl CoAs with the MS operated in positive mode, as previous studies demonstrated that they are more efficiently ionized under these conditions [[15]15].. We We observed the characteristiccharacteristic fragmentation pattern for all acylacyl CoA species. The CoA portion was cleaved at the 33′0--phosphate-adenosine-5phosphate-adenosine-5′0-diphosphate-diphosphate during positive mode MS/MS.MS/MS. This This fragmentation fragmentation gave rise to a neutral loss of 507 atomic mass units (amu) (amu) together together with with a a daughter daughter ion ion equal equal to to a mass a mass-to-charge-to-charge ratio ratio (m/z) (m/z) of [M of + [M− 507− +507 H] +, H]where+, where “M” “M”is the is molecular the molecular mass mass of the of initial the initial compound. compound. Additionally, Additionally, the thephosphate phosphate-adenosine-adenosine portion portion of each of acyl each CoA acyl species CoA species fragmented fragmented between between the 5′-diphos- the 50- diphosphatesphates and gave and rise gave to risea daughter to a daughter equal to equal 428 tom/z 428 [26] m/z (Figure [26] (Figure 2A). For2A). example, For example, acetyl acetylCoA has CoA a monoisotopic has a monoisotopic mass of mass 809.1 of and 809.1 therefore and therefore gives rise gives to rise an MS1 to an parent MS1 parent of 810.1 of 810.1m/z. Following m/z. Following fragmentation, fragmentation, daughter daughter ions ionsof 303 of 303m/z m/z(indicating (indicating cleavage cleavage at the at the3′- 3phosphate0-phosphate-adenosine-5-adenosine-5′-diphosphate)0-diphosphate) and and 428 428 m/z m/z (indicating (indicating fragmentation fragmentation between between the the5′ diphosphates) 50 diphosphates) were were both both apparent apparent (Figure (Figure 2B,C).2B,C). We We used used multiple multiple reaction reaction monitoring monitoring(MRM) (MRM) to both to both identify identify and and quantify quantify each each acyl acyl CoA CoA species species detected detected with with this this method. method. For each acyl CoA,CoA, twotwo separateseparate transitionstransitions werewere monitored:monitored: (i)(i) [M[M ++ H]H]++ fragmentingfragmenting toto [M − 507507 + + H] H]+ m/z+ m/z was was used used for for quantitation, quantitation, and and (ii) (ii)[M [M+ H] ++ H] fragmenting+ fragmenting to 428 to 428m/z m/z was wasused used for qualitative for qualitative identification identiﬁcation (Table (Table 1). 1).

Parent: [M+H]+ m/z A N OH O O O H H R N N N H2N O P O P O S

O N N OH OH O HO O

HO P OH

O Daughter 1: 428 m/z Daughter 2: [M-507+H]+ m/z N O OH N H N 2 OH + O P OH2 H H R + N N H2C N N O S HO O

O O HO P OH

O B Parent: 810 m/z N OH O O O O H H N N N H2N O P O P O S

O N N OH OH O HO O

HO P OH

O Daughter 1: 428 m/z Daughter 2: 303 m/z N O OH N H N 2 O + OH O P OH2 H H + N N N N H2C O S HO O

O O HO P OH

O C

Figure 2. The commoncommon MS/MSMS/MS fragmentationfragmentation pattern pattern for for all all CoA CoA species: species: (A ()A The) The CoA CoA portion portion of allof CoAall CoA esters esters fragments fragments during during MS/MS MS/MS at theat the 30-phosphate-adenosine-5 3′-phosphate-adenosine0-diphosphate.-5′-diphosphate. This This cleavage cleav- gaveage gave rise rise to a to daughter a daughter ion ion equal equal to [M to [M− 507 − 507 + H]+ H]+ m/z+ m/z (right). (right). “M”“M” isis thethe molecularmolecular massmass ofof thethe starting compound. The phosphate-adenosine portion also fragments between the 50-diphosphates and gave rise to a daughter equal to 428 m/z (left). (B) Acetyl CoA has a parent m/z of 810 with a predicted fragmentation pattern for daughter ions of 428 m/z and 303 m/z. (C) The MS1 parent and predicted MS2 daughter ions for acetyl CoA were detected following direct infusion of 1 µg/mL acetyl CoA into the mass spectrometer. Metabolites 2021, 11, x FOR PEER REVIEW 5 of 19

Metabolites 2021, 11, x FOR PEER REVIEW 5 of 19

starting compound. The phosphate-adenosine portion also fragments between the 5′-diphosphates Metabolites 2021, 11, x FOR PEER REVIEWand gave rise to a daughter equal to 428 m/z (left). (B) Acetyl CoA has a parent m/z of 810 5with of 19 a predicted fragmentation pattern for daughter ions of 428 m/z and 303 m/z. (C) The MS1 parent and starting compound. The phosphate-adenosine portion also fragments between the 5′-diphosphates predicted MS2 daughter ions for acetyl CoA were detected following direct infusion of 1 μg/mL and gave rise to a daughter equal to 428 m/z (left). (B) Acetyl CoA has a parent m/z of 810 with a acetyl CoA into the mass spectrometer. Metabolites 2021, 11, x FOR PEER REVIEWstapredictedrting compound. fragmentation The phosphate pattern for-adenosine daughter portion ions of 428also m/z fragments and 303 between m/z. (C )the The 5′ MS1-diphosphates parent5 of and 19 predicted MS2 daughter ions for acetyl CoA were detected following direct infusion of 1 μg/mL Table 1. Parent and daughtergave rise ionto a m/z daughter for short equal-chain to 428acyl m/z CoAs, (left). dephospho (B) Acetyl-CoA, CoA and has pantothen a parent m/zate. of 810 with a predictedacetyl CoA fragmentation into the mass pattern spectrometer. for daughter ions of 428 m/z and 303 m/z. (C) The MS1 parent and Compound Acylpredicted Group MS2 daughterAcyl ions Group for acetyl CoA were detected followingDaughter direct 1 infusion Daughter of 1 μg/mL 2 Metabolites 2021, 11, x FOR PEER REVIEWstarting compound. The phosphate-adenosineParent portion (m/z) also fragments between the 5′-diphosphates5 of 19 Name Table 1. ParentFormula andacetyl daughter CoA into ion the m/z mass forStructure spectrometer.short-chain acyl CoAs, dephospho-CoA, and(m/z) pantothen ate. (m/z) and gave rise to a daughter equal to 428 m/z (left). (B) Acetyl CoA has a parent m/z of 810 with a CompoundCoA Acyl HGroup Acyl HGroup 768.1 Daughter261.1 1 Daughter428.1 2 Table 1. Parent andpredicted daughter fragmentation ion m/z for short pattern-chain for acyl daughter CoAs, ionsdephosphoParent of 428 (m/z) m/z-CoA, and and 303 pantothen m/z. (C) Theate. MS1 parent and Metabolites 2021, 11, x FOR PEER REVIEWpredicted MS2 daughter ions for acetyl CoA were detected following direct infusion of 1 μg/mL5 of 19 Name Formulastarting compound. TheStructure phosphate -adenosine portion also fragments(m/z) between the 5′-diphosphates(m/z) Compound Acyl Groupacetyl CoA into the massAcyl spectrometer. Group Daughter 1 Daughter 2 CoA andH gave rise to a daughterH equal to 428 m/zParent (left).768.1 ((m/z)B) Acetyl CoA 261.1has a parent m/z of428.1 810 with a Acetyl CoA COCH3 810.1 303.1 428.1 MetabolitesName 2021, 11, x FOR PEERFormula REVIEWpredicted fragmentationStructure pattern for daughter ions of 428 m/z and 3(m/z)03 m/z. (C) The MS1(m/z) parent 5 of and 19 Table 1. Parent and daughter ion m/z for short-chain acyl CoAs, dephospho-CoA, and pantothenate. CoA Hstapredicted rting compound. MS2 daughter The phosphate ionsH for acetyl- adenosine CoA portionwere768.1 detected also fragments following261.1 between direct infusionthe 5′-diphosphates428.1 of 1 μg/mL acetyl CoA into the mass spectrometer. CompoundAcetyl CoA AcylCOCH andGroup gave3 rise to a daughterAcyl Groupequal to 428 m/z (left).810.1 (B) Acetyl CoADaughter has303.1 a parent 1 m/zDaughter of428.1 810 with 2 a predicted fragmentation pattern for daughter ionsParent of 428 (m/z) m/z and 303 m/z. (C) The MS1 parent and Name Table 1. ParentFormula andstarting daughter compound. ion m/z The forStructure phosphateshort-chain - adenosine acyl CoAs, portion dephospho also fragments-CoA, and(m/z) betweenpantothen theate. 5′ -diphosphates(m/z) Acetyl CoA COCHpredicted3 MS2 daughter ions for acetyl CoA were810.1 detected following303.1 direct infusion428.1 of 1 μg/mL CoA andacetylH gave CoA rise into to thea daughter mass spectrometer. Hequal to 428 m/z (left).768.1 (B) Acetyl CoA has261.1 a parent m/z of428.1 810 with a PropionylCompound CoA COCHAcylpredicted Group2CH3 fragmentationAcyl pattern Group for daughter ions of824.1 428 m/z andDaughter 303317.1 m/z. (C 1) The MS1Daughter428.1 parent and2 Parent (m/z) Name Formulapredicted MS2 daughterStructure ions for acetyl CoA were detected following(m/z) direct infusion of(m/z) 1 μg/mL Metabolites 2021Table, 11 1., 468 Parent and daughter ion m/z for short-chain acyl CoAs, dephospho-CoA, and pantothenate. 5 of 18 acetyl CoA into the mass spectrometer. PropionylAcetylCoA CoA CoA COCHCOCHH 2CH3 3 H 810.1824.1768.1 303.1317.1261.1 428.1428.1 Compound Acyl Group Acyl Group Daughter 1 Daughter 2

Table 1. Parent and daughter ion m/z for short-chain acyl CoAs,Parent dephospho (m/z)-CoA, and pantothenate. PropionylName CoA COCHFormula2CH3 Structure 824.1 317.1(m/z) 428.1(m/z) IsovalerylCompoundAcetylCoA CoA CoA TableCOCH 1.AcylParentCOCH HGroup2(CH and3 3) daughter2 ion m/zAcyl for HGroup short-chain acyl CoAs,768.1852.1810.1 dephospho-CoA, Daughter261.1345.1303.1 and pantothenate.1 Daughter428.1428.1428.1 2 Parent (m/z) NameCompound FormulaAcyl Group StructureAcyl Group (m/z)Daughter 1 (m/z)Daughter 2 Parent (m/z) PropionylIsovalerylCoAName CoACoA COCHCOCHHFormula2 (CH2CH3) 2 StructureH 768.1824.1852.1 261.1317.1345.1(m/z) 428.1428.1428.1 (m/z) Acetyl CoA COCH3 810.1 303.1 428.1 CoA H H 768.1 261.1 428.1 Isovaleryl CoA COCH2(CH3)2 852.1 345.1 428.1 PropionylMalonylAcetylAcetyl CoA CoA CoACoA COCHCOCHCOCH COCH2CO2CH3 2H33 810.1810.1854.1824.1 303.1347.1317.1 303.1 428.1428.1428.1 428.1

Malonyl CoA COCH2CO2H 854.1 347.1 428.1 IsovalerylPropionyl CoA CoA COCH COCH2(CH3CH)2 852.1824.1 345.1 317.1 428.1428.1 Propionyl CoA COCH2CH23 3 824.1 317.1 428.1

Malonyl CoA COCH2CO2H 854.1 347.1 428.1 Isovaleryl CoA COCH (CH ) 852.1 345.1 428.1 PropionylIsovaleryl CoA CoA COCHCOCH22(CHCH2 33 )2 3 2 824.1852.1 317.1345.1 428.1428.1 Succinyl CoA CO(CH2)2CO2H 868.1 361.1 428.1

Malonyl CoA COCH CO H 854.1 347.1 428.1 Malonyl CoA COCH2CO22H 2 854.1 347.1 428.1 IsovalerylSuccinyl CoACoA CO(CHCOCH22(CH)2CO3)22H 852.1868.1 345.1361.1 428.1428.1 Compoud Compound Daughter 1 Daughter 2 SuccinylSuccinyl CoA CoAChemicalCO(CH CO(CH2)2 COFormula)2HCO H Parent868.1868.1 (m/z) 361.1 361.1 428.1 428.1 IsovalerylMalonylName CoA COCHCOCH2(CH2CO2 32)H2 2 Structure 852.1854.1 345.1(m/z)347.1 428.1(m/z)428.1 Compoud Compound Daughter 1 Daughter 2 Chemical Formula Parent (m/z) NameCompoud Chemical CompoundStructure (m/z)Daughter 1 (m/z)Daughter 2 Pantothenate C9H172NO25 Parent218.0 (m/z) 88.0 145.8 MalonylSuccinylCompoudName CoA CO(CHCOCHFormula2)CO2COH2H CompoundStructure 854.1868.1 Daughter347.1361.1(m/z) 1 Daughter428.1(m/z) 2 Chemical Formula Parent (m/z) Name Structure (m/z) (m/z) PantothenatePantothenate C9H C17NOH NO5 218.0218.0 88.0 88.0 145.8 145.8 Malonyl CoA COCH29CO172H 5 854.1 347.1 428.1 Succinyl CoA CO(CH2)2CO2H 868.1 361.1 428.1 Compoud Compound Daughter 1 Daughter 2 21 35 7 13 2 DephosphoPantothenateDephospho-CoA-CoA ChemicalCCH9 CH2117NHNO 35FormulaON5 7PO13S P2 S Parent218.0688.1 (m/z) 88.0261.1 261.1 145.8348.1 348.1 Name Structure (m/z) (m/z) Succinyl CoA CO(CH2)2CO2H 868.1 361.1 428.1 DephosphoCompoud-CoA C21H35N7O13P2S Compound 688.1 Daughter261.1 1 Daughter348.1 2 Chemical FormulaAfter tuning for efﬁcient detection Parent of acyl (m/z) CoAs, we optimized the detection of CoA PantothenateName C9H17NOAfter5 tuning for Structureefficient detection of acyl218.0 CoAs, we optimized(m/z)88.0 the detection145.8(m/z) of CoA

DephosphoSuccinyl CoA-CoA CCO(CH21H35Nsynthetic27)O2CO13Psynthetic22SH intermediates. intermediates. Dephospho Dephospho-CoA,-CoA, similar688.1868.1 similar to acyl to acyl CoAs,261.1361.1 CoAs, was was efficiently efficiently348.1428.1 detected detected in positive mode. The quantitative and qualitative MRM transitions of dephospho-CoA were Compoud in positive mode. TheCompound quantitative and qualitative MRM Daughtertransitions 1 of dephosphoDaughter- CoA2 Chemical FormuladeterminedAfter tuning empirically for efficient during detection tuning ofParent acyl (Table CoAs, (m/z)1). Initial we optimized efforts to optimizethe detection the detectionof CoA of PantothenateName C9Hwere17NO determined5 empiricallyStructure during tuning (Table218.0 1). Initial efforts(m/z)88.0 to optimize(m/z)145.8 the detec- syntheticpantothenate intermediates. in the positiveDephospho mode -CoA, MRM similar were to unsuccessful, acyl CoAs, andwas muchefficiently greater detected sensitivity Dephospho-CoA C21H35N7O13P2S 688.1 261.1 348.1 Compoud in positiveAfterwas observedtuning mode. for The usingCompound efficient quantitative the negativedetection and modeof qualitative acyl MRM CoAs, (Figure MRM we optimizedDaughter transitions S2). As athe 1 result, of detection dephosphoDaughter the negative of CoA- CoA2 mode Chemical Formula Parent (m/z) Name syntheticwereMRM determined intermediates. was used empiricallyStructure for Dephospho pantothenate, during -CoA, tuning while similar the(Table positive to 1). acyl Initial mode CoAs, (m/z)efforts MRM was to wasefficiently optimize used(m/z) for detectedthe all detec- other ana- Pantothenate C9H17NO5 218.0 88.0 145.8 in positivelytes. mode. Additional The settingsquantitative for each and transition qualitative including MRM transitions declustering of potentialdephospho (DP),-CoA entrance Dephospho-CoA C21H35wereN7O determined13P2S empirically during tuning (Table688.1 1). Initial efforts261.1 to optimize the348.1 detec- potentialAfter tuning (EP), for collision efficient energy detection (CE), of andacyl cell CoAs, exit we potential optimized (CXP) the were detection optimized of CoA during Pantothenate C9H17NO5 218.0 88.0 145.8 synthetictuning intermediates. with reference Dephospho standards -CoA, (Supplemental similar to Tableacyl CoAs, S1). Source was efficiently settings of detected temperature, in positiveion spray mode. voltage, The quantitative and gas flows and were qualitative likewise MRM optimized transitions during of methoddephospho development-CoA Dephospho-CoA C21H35N7O13P2S 688.1 261.1 348.1 were(see Afterdetermined Methods). tuning empirically for efficient during detection tuning of acyl (Table CoAs, 1). Initialwe optimized efforts to the optimize detection the of detec- CoA synthetic intermediates. Dephospho-CoA, similar to acyl CoAs, was efficiently detected 2.2. Ion-Pairing UHPLC Chromatography Produces Well-Separated Peaks for CoA Biosynthetic Dephospho-CoA C21H35Nin7O positive13P2S mode. The quantitative and qualitative688.1 MRM transitions261.1 of dephospho348.1 -CoA Intermediates and Short-Chain Acyl CoAs wereAfter determined tuning forempirically efficient detectionduring tuning of acyl (Table CoAs, 1). we Initial optimized efforts tothe optimize detection the of detec- CoA synthetic Weintermediates. next developed Dephospho the chromatography-CoA, similar to parameters acyl CoAs, for was CoA efficiently biosynthetic detected interme- in positivediates mode. and short-chain The quantitative acyl CoAs. and qualitative Like other MRM phosphorylated transitions organicof dephospho molecules,-CoA CoA After tuning for efficient detection of acyl CoAs, we optimized the detection of CoA werecan determined be a difficult empirically structure during to resolve tuning using(Table common 1). Initial chromatographicefforts to optimize techniques the detec- [25]. syntheticIts three intermediates. phosphate Dephospho groups are-CoA, polar similar and capable to acyl of CoAs, negatively was efficiently ionizing atdetected higher pH, in positiveresulting mode. in poor The affinityquantitative for the and solid qualitative phase of MRM common transitions reverse of phase dephospho columns-CoA like C18. were Simultaneously,determined empirically the adenine during moiety tuning can (Table serve 1). as Initial a Bronsted–Lowry efforts to optimize base the and detec- acquire a proton at a pH below 4.0. Moreover, these polar or charged groups are offset by acyl groups of increasing non-polarity as the length of the acyl chain increases. We first tested a standard HPLC C18 column using an acetonitrile gradient containing common modifiers such as 0.1% (v/v) formic acid or 5 mM ammonium acetate. Formic

acid produced extremely poor chromatography (not shown), thus ammonium acetate was used as the modiﬁer and proton source. Unmodiﬁed ammonium acetate still produced considerable peak tailing, thus the pH was carefully adjusted with acetic acid to pH 5.6. Metabolites 2021, 11, 468 6 of 18

+ This value was selected in order to be above the pKa of the adenine NH3 (4.0) but below the pKa of the secondary phosphate (6.4) [27]. While the chromatography of the CoA species was improved, it still exhibited poor peak shape. The poor results at pH 5.6 are likely explained by the primary phosphate of the CoA backbone retaining a negative charge and pantothenate remaining as an anion. The remaining chromatographic difﬁculties were resolved by ion pairing chromatography combined with use of a UHPLC C18 column with high theoretical plates. N,N- dimethylbutylamine (DMBA) is a tertiary amine that acquires a proton below pH 10.0 [28] and has been used as an ion pairing agent to improve the chromatography of phosphate- containing compounds such as oligonucleotides [29]. DMBA has also been used to improve the chromatography of short-chain acyl CoA species such as malonyl CoA [17]. It was therefore added to the ammonium acetate solution (solvent A; Table2) and a Phenomenex Kinetex ultra-high performance liquid chromatography (UHPLC) C18 column was used. Metabolites 2021, 11, x FOR PEER REVIEW 7 of 19 The resulting chromatography produced well-separated peaks with minimal tailing for both CoA biosynthetic intermediates and short chain acyl CoAs (Figure3).

Figure 3.Figure Ion-pairing 3. Ion-pairing UHPLC chromatography UHPLC chromatography produces well produces-separated well-separated peaks for CoA peaks biosynthetic for CoA intermediates biosynthetic and short-chainintermediates acyl CoAs. All and eight short-chain compounds acyl together CoAs. with All the eight internal compounds standard together (crotonoyl with CoA) the were internal injected standard at 1 μg/mL into the (crotonoylLC-MS/MS. CoA) Retention were times injected are listed at 1 µ g/mLto the right into theof each LC-MS/MS. peak. Liquid Retention chromatography times are consisted listed to of the a rightreverse- phase gradient with solvents A & B (see Table 2) in addition to a Phenomenex Kinetex, 2.6 mm × 150 mm UHPLC C18 column.of The each quantitative peak. Liquid MRM channel chromatography of each species consisted was isolated of a reverse-phaseand retention times gradient were determined. with solvents A & B (see Table2) in addition to a Phenomenex Kinetex, 2.6 mm × 150 mm UHPLC C18 column. The quantitative MRMTable channel 2. The of following each species solvents was were isolated used and during retention the chromatography: times were determined. Solvent A, 5 mM ammonium acetate + 2.5 mM DMBA (pH 5.6) and Solvent B, 95% acetonitrile, 5% H2O + 5 mM ammonium Importantly,acetate. dephospho-CoA appears to form in the electrospray ionization (ESI) source from acetyl, propionyl,Time (min and) isovaleryl%ACoA %B (Figure S3). It is thereforeNotes crucial to carefully distinguish the peaks0 and retention98 times associated2 with dephospho-CoADivert to waste itself from those resulting from these1.5 other analytes98 in order2 to avoid erroneous(0–5.5 detectionmin) of dephospho-CoA. 9 50 50 Divert to MS 9.5 2 98 (5.5–10.5 min) 14.5 2 98 Divert to waste 15 98 2 (10.5–22 min) 22 98 2

Importantly, dephospho-CoA appears to form in the electrospray ionization (ESI) source from acetyl, propionyl, and isovaleryl CoA (Figure S3). It is therefore crucial to carefully distinguish the peaks and retention times associated with dephospho-CoA itself from those resulting from these other analytes in order to avoid erroneous detection of dephospho-CoA.

2.3. The Method Displays a Linear Detection of Analytes Across a Wide Concentration Range with Sensitive Lower Limits of Detection and Quantitation The sensitivity and linearity of the LC-MS/MS method were determined by spiking a range of reference analytes into the sample extraction solution in addition to the internal

Metabolites 2021, 11, 468 7 of 18

Table 2. The following solvents were used during the chromatography: Solvent A, 5 mM ammonium

acetate + 2.5 mM DMBA (pH 5.6) and Solvent B, 95% acetonitrile, 5% H2O + 5 mM ammonium acetate.

Time (min) %A %B Notes 0 98 2 Divert to waste 1.5 98 2 (0–5.5 min) 9 50 50 Divert to MS 9.5 2 98 (5.5–10.5 min) 14.5 2 98 Divert to waste 15 98 2 (10.5–22 min) 22 98 2

2.3. The Method Displays a Linear Detection of Analytes across a Wide Concentration Range with Metabolites 2021, 11, x FOR PEER REVIEWSensitive Lower Limits of Detection and Quantitation 8 of 19

The sensitivity and linearity of the LC-MS/MS method were determined by spiking a range of reference analytes into the sample extraction solution in addition to the internal standard crotonoyl crotonoyl CoA. CoA. Calibration Calibration curves curves were ﬁtwere by linear fit by regression linear regression with 1/x weighting with 1/x (Figure4). Figure 4 weighting (Figure 4).

)

d 10 Pantothenate

a Dephospho-CoA

n CoA

a t 8 Malonyl CoA

l Succinyl CoA

n Acetyl CoA

e Propionyl CoA t 6

i Isovaleryl CoA

e 4

o 2

(

A 0 0 500 1000 pmol analyte

FigureFigure 4. The LC-MS/MSLC-MS/MS method method demonstrates demonstrates a a linear linear detection detection of analytes across a wide con- concen- trationcentration range: range: standards standards were were spiked spiked into into 200 µ200L of μL extraction of extraction solution solution and analyzedand analyzed via LC-MS/MS. via LC- CalibrationMS/MS. Calibration curves were curves generated were generated by plotting by plotting the area the under area under the curve the cur (AUC)ve (AUC) for each for each analyte (normalizedanalyte (normalized to the internal to the standard)internal standard) against the against amount the of amount analyte of contained analyte contained in 200 µL ofin extraction200 μL of extraction solution. Data are presented as mean  standard error of the mean (S.E.M.) for n  3 solution. Data are presented as mean ± standard error of the mean (S.E.M.) for n ≥ 3 technical technical replicates. replicates.

The lower limit of detectiondetection (LLOD)(LLOD) forfor eacheach analyteanalyte waswas determineddetermined asas thethe lowestlowest concentration thatthat produced aa signal at least five ﬁve times greater than that of the the correspond- correspond- ing noise noise floor. ﬂoor. The The lower lower limit limit of ofquantitation quantitation (LLOQ) (LLOQ) was was determined determined as the as lowest the lowest con- concentrationcentration in tinhe the calibration calibration curve curve for for which which the the curvecurve-calculated-calculated concentration was was withinwithin 20%20% ofof itsits nominalnominal concentration. concentration. The The linearity linearity of of each each calibration calibration curve curve was was deter- de- termined as the coefficient of correlation r. For each analyte, the LC-MS/MS yielded sensitive LLOD and LLOQ with r ≥ 0.95 (Table 3).

Table 3. The lower limits of detection (LLOD) and quantitation (LLOQ) are calculated as described in the text.

Compound Linear Regression r LLOD (pmol) LLOQ (pmol) Pantothenate Y = 0.00264x + 0.0307 0.98 1 7.4 Dephospho-CoA Y = 0.00481x + 0.0892 0.97 0.4 3.7 CoA Y = 0.00546x − 0.0196 0.97 1 3.7 Malonyl CoA Y = 0.00221x − 0.00851 0.99 3 3.7 Succinyl CoA Y = 0.00095x − 0.0011 0.95 1 7.4 Acetyl CoA Y = 0.00461x − 0.0163 0.97 1 3.7 Propionyl CoA Y = 0.00660x + 0.0367 0.99 2 3.7 Isovaleryl CoA Y = 0.00864x − 0.1853 0.99 1 7.4

Metabolites 2021, 11, 468 8 of 18

mined as the coefﬁcient of correlation r. For each analyte, the LC-MS/MS yielded sensitive LLOD and LLOQ with r ≥ 0.95 (Table3).

Table 3. The lower limits of detection (LLOD) and quantitation (LLOQ) are calculated as described in the text.

2.4. Extraction with 2.5% SSA Is Suitable for Analysis of Acyl CoAs and CoA Biosynthetic Intermediates Following refinement of the LC-MS/MS settings, we next determined an appropriate method to extract acyl CoAs and CoA biosynthetic intermediates from biological material. We first evaluated common acyl CoA extraction procedures to test their capacity to recover dephospho-CoA and pantothenate. Acyl CoAs are commonly extracted with halogenated forms of acetic acid [e.g., trichloroacetic acid (TCA) and trifluoroacetic acid (TFA)] followed by solid phase extraction (SPE) to remove the deproteinizing agent. After SPE purification, acyl CoAs are resuspended in a solvent favorable for LC-MS/MS analysis such as water or SSA [18,19,22]. To control for variabilities in the sample extraction procedure, we again used crotonoyl CoA as an internal standard. Complete precision under all possible conditions for a given CoA species or biosynthetic intermediate requires a matched, isotopically labeled reference species. This can be accomplished by SILEC labeling [21], which exploits the lack of de novo pantothenate synthesis in mammalian and insect cells to generate such standards with the provision of labeled pantothenate in cell culture medium. Although this technique is the gold standard for such assays, it can be time and cost prohibitive for almost all non-specialist labs. We therefore used crotonyl CoA as an inexpensive and amenable standard for this straightforward method. Figure S4 illustrates that endogenous crotonyl CoA levels range from less than 1% (cultured cells) to an upper bound of 3% (5 mg liver tissue) of the internal standard. As such, endogenous changes in crotonyl CoA levels would only introduce a marginal error when quantifying short-chain CoAs and CoA biosynthetic intermediates under most conditions. To test whether TCA followed by SPE was a suitable extraction technique, 1 nmol of standards for pantothenate, dephospho-CoA, and each acyl CoA were extracted with 200 µL of 10% (w/v) TCA. TCA was removed by SPE and samples were reconstituted in 2.5% SSA prior to LC-MS/MS analysis [16,21,22]. For comparison, 1 nmol of each standard was also spiked into 200 µL of water and similarly analyzed. Relative recovery was determined by comparing the area under the curve (AUC) of each analyte extracted with TCA with the analyte in water. While using TCA demonstrated moderate recovery of acetyl CoA and CoA species with longer acyl tails, there was a marked decrease in recovery of free CoA and the biosynthetic intermediates (Figure5A). Metabolites 2021, 11, x FOR PEER REVIEW 9 of 19

2.4. Extraction with 2.5% SSA Is Suitable for Analysis of Acyl CoAs and CoA Biosynthetic Intermediates Following refinement of the LC-MS/MS settings, we next determined an appropriate method to extract acyl CoAs and CoA biosynthetic intermediates from biological material. We first evaluated common acyl CoA extraction procedures to test their capacity to recover dephospho-CoA and pantothenate. Acyl CoAs are commonly extracted with halogenated forms of acetic acid [e.g., trichloroacetic acid (TCA) and trifluoroacetic acid (TFA)] followed by solid phase extraction (SPE) to remove the deproteinizing agent. After SPE purification, acyl CoAs are resuspended in a solvent favorable for LC-MS/MS analysis such as water or SSA [18,19,22]. To control for variabilities in the sample extraction procedure, we again used crotonoyl CoA as an internal standard. Complete precision under all possible conditions for a given CoA species or biosynthetic intermediate requires a matched, isotopically labeled reference species. This can be accomplished by SILEC labeling [21], which exploits the lack of de novo pantothenate synthesis in mammalian and insect cells to generate such standards with the provision of labeled pantothenate in cell culture medium. Although this technique is the gold standard for such assays, it can be time and cost prohibitive for almost all non-specialist labs. We therefore used crotonyl CoA as an inexpensive and amenable standard for this straightforward method. Figure S4 illustrates that endogenous crotonyl CoA levels range from less than 1% (cultured cells) to an upper bound of 3% (5 mg liver tissue) of the internal standard. As such, endogenous changes in crotonyl CoA levels would only introduce a marginal error when quantifying short-chain CoAs and CoA biosynthetic intermediates under most conditions. To test whether TCA followed by SPE was a suitable extraction technique, 1 nmol of standards for pantothenate, dephospho-CoA, and each acyl CoA were extracted with 200 μL of 10% (w/v) TCA. TCA was removed by SPE and samples were reconstituted in 2.5% SSA prior to LC-MS/MS analysis [16,21,22]. For comparison, 1 nmol of each standard was also spiked into 200 μL of water and similarly analyzed. Relative recovery was determined by comparing the area under the curve (AUC) of each analyte extracted with TCA

Metabolites 2021, 11, 468 with the analyte in water. While using TCA demonstrated moderate recovery of acetyl9 of 18 CoA and CoA species with longer acyl tails, there was a marked decrease in recovery of free CoA and the biosynthetic intermediates (Figure 5A).

7 3000 A 3×10 Water B

)

7 s l 2500 2×10 10% TCA l TCA

) 7 c 1×10 2.5% SSA

6 C 2000 SSA

A 6 x 1500 ( 4×10

l 50

c 6

n 3×10 m 40

(

e n 30

6 c

u 2×10

d A 20 6 1×10 n

b 10 0 A 0 te A A A A A A a o o o o o o e n C C C C C C t A A A e - l l l l a o o o h o y y y y n C C C t h n t n r e l l to p o e o le th ty y s l c i a e n n o a A p v to c o a h M o o n i P p r s a A p e P I P ro D P FigureFigure 5.5. SSA-basedSSA-based sample preparation results in higher recovery of CoA biosynthetic pathway intermediates and short short-- chainchain acylacyl CoAs.CoAs. ((AA)) 1 1 nmol nmol of of each each analyte analyte was was extracted extracted with with 200 200µL μL of 10%of 10% (w/ (vw)/ TCAv) TCA followed followed by solid-phaseby solid-phase extraction, extraction, 2.5% (w/v) SSA, or spiked into 200μL water. The following percent recoveries were obtained relative to water and 2.5% (w/v) SSA, or spiked into 200µL water. The following percent recoveries were obtained relative to water and shown as shown as TCA vs. SSA: pantothenate (0% vs. >100%); dephospho-CoA (0% vs. >99%); CoA (1% vs. 74%); malonyl CoA TCA vs. SSA: pantothenate (0% vs. >100%); dephospho-CoA (0% vs. >99%); CoA (1% vs. 74%); malonyl CoA (26% vs. (26% vs. 74%); acetyl CoA (36% vs. 59%); propionyl CoA (62% vs. 80%); and isovaleryl CoA (58% vs. 59%). (B) Metabolites 74%);were extracted acetyl CoA from (36% HEK vs. 293FT 59%); propionylcells using CoAeither (62% direct vs. extraction 80%); and with isovaleryl SSA or CoA TCA (58% followed vs. 59%). by solid (B)- Metabolitesphase extraction. were extractedAll data are from presented HEK 293FT as mean cells using standard either error direct of extraction the mean with(S.E.M.) SSA for or TCAn  3 followedtechnical byreplicates. solid-phase extraction. All data are presented as mean ± standard error of the mean (S.E.M.) for n ≥ 3 technical replicates.

In the search for alternatives, we noted that SSA, in addition to being a commonly

used solvent for reconstituting acyl CoAs for LC-MS/MS [16,21,22], has also been used for deproteinizing biological samples [30,31]. We determined that 2.5% (w/v) SSA fully deproteinized biological samples without lowering the pH of the extraction solution below 1.0, the recommended lower threshold for the HPLC column used in this study. Indeed, the extraction with 2.5% SSA exhibited a similar recovery of hydrophobic short-chain acyl CoAs relative to TCA. Additionally, we observed an increased recovery of free CoA and the CoA biosynthetic precursors of pantothenate and dephospho-CoA (Figure5A). In terms of SSA, 2.5% was also able to efﬁciently extract these metabolites from the HEK 293FT human embryonic kidney cell line and compared favorably to TCA (Figure5B). The results demonstrate that SSA is a preferred extraction solvent for our optimized LC-MS/MS method and obviates the need for solid phase extraction for these metabolites.

2.5. The Sample Preparation and LC-MS/MS Method Demonstrates the Minimal Matrix Effect and Preserves Accuracy Having demonstrated that 2.5% SSA is a suitable extraction solvent, we then determined if our method displayed minimal variability due to matrix effects across the range of analytes. A matrix effect was determined as the percentage ratio of the AUC of analyte that had been spiked into the matrix versus the LC-MS/MS AUC of analyte that had been spiked into the extraction solution alone (Supplemental Table S2). For each analyte apart from dephospho-CoA, the matrix effect was marginal and exhibited less than a 10% ion suppression. Dephospho-CoA exhibited a consistently greater matrix effect, with an ion suppression of an average of 19% across its low, medium, and high spikes into the matrix. Comparative accuracy was determined as the percent ratio of the mean baseline- subtracted concentrations for the spikes into the post-extraction matrix compared to mean concentrations of spikes into the extraction solution alone. In general, the internal standard- corrected matrix effect was deemed acceptable if it produced comparative accuracy within 85%. All analytes including dephospho-CoA achieved this average comparative accuracy. As such, despite the relatively large matrix effect of dephospho-CoA, this was partially compensated for by the internal standard when determining the concentration. Metabolites 2021, 11, 468 10 of 18

2.6. The Method Exhibits Acceptable Precision and Accuracy Parameters for Measured Analytes Across Their Entire Linear Ranges The precision and accuracy for the sample preparation and LC-MS/MS method were determined according to guidelines provided by the FDA [32] and EMA [33] (Table4). Low, medium, and high amounts of eight analytes were added to cell pellets, and the concentration was determined. Precision was reported as the relative standard deviation or coefﬁcient of variation (CV). In general, precision was deemed acceptable with 15% ≥ CV and all metabolites considered ﬁt this criterion. Accuracy was determined by comparing the average observed concentration to the amount spiked at that concentration after baseline correction. Accuracy was deemed acceptable when mean values were within 15% of the nominal values, and all metabolites considered met this mark.

Table 4. The precision and accuracy are determined as described in the text. NA is deﬁned as not applicable.

Calculated Accuracy Precision Standards (pmol) Concentration SEM (% of Nominal CV (%) (pmol/1 × 106 cells) Value) Pantothenate 0 969.15 7.74 1.60 NA 62.5 1231.50 48.44 7.87 119.37 250 1421.00 78.83 11.10 116.56 1000 2244.50 31.47 2.80 113.98 Dephospho-CoA 0 1.68 0.08 9.78 NA 62.5 57.39 4.78 16.64 89.41 250 228.81 23.02 20.12 90.91 1000 961.96 24.63 5.12 96.03 CoA 0 49.98 4.67 18.68 NA 62.5 143.03 4.81 6.73 127.16 250 277.50 16.61 11.97 92.51 1000 865.48 7.02 1.62 82.43 Acetyl CoA 0 65.67 1.81 5.52 NA 62.5 127.05 8.85 13.94 99.13 250 356.48 14.96 8.40 112.93 1000 1402.79 32.15 4.58 131.63 Propionyl CoA 0 7.95 0.20 5.03 NA 62.5 75.03 5.98 15.93 106.49 250 277.52 4.06 2.92 107.59 1000 1132.35 59.58 10.52 112.34 Isovaleryl CoA 0 3.17 0.21 13.19 NA 62.5 67.53 6.33 18.76 102.82 250 314.52 4.34 2.76 124.23 1000 1383.19 50.47 7.30 137.88 Malonyl CoA 0 2.45 0.18 14.84 NA 62.5 62.03 2.94 9.48 95.50 250 265.98 20.15 15.15 105.36 1000 1077.85 69.21 12.84 107.52 Succinyl CoA 0 208.61 11.79 11.31 NA 62.5 295.38 22.62 15.32 108.96 250 507.42 25.24 9.95 110.64 1000 1279.86 71.49 11.17 105.90

2.7. The Method Detects Expected Perturbations in Levels of CoA and CoA Esters We next validated our method by assessing acyl CoA levels in cells following treat- ments previously shown to increase and decrease steady-state levels of CoA-related metabolites. To determine if we could detect an enhanced synthesis of speciﬁc acyl CoAs, we treated HepG2 cells with 1 mM propionate. Consistent with prior reports [16], we observed Metabolites 2021, 11, 468 11 of 18

a more than ten-fold increase in propionyl CoA levels following treatment (Figure6A). We then measured whether the method could detect an increase in CoA synthesis with PZ-2891, a pantazine compound that is effective in treating preclinical models of pantothenate kinase-associated neurodegeneration (PKAN) [34]. The drug increases CoA levels by stabilizing pantothenate kinase in its active conformation. Indeed, HepG2 cells treated Metabolites 2021, 11, x FOR PEER REVIEW 12 of 19 with 10 µM PZ-2981 caused an expected two-fold increase in free CoA in addition to an increased abundance of both acetyl and propionyl CoA (Figure6B).

)

A B ) A B )

s 100

s s 100

l VVeehhicilcle e 80

l l VVeehhicilcle e 80

Propionate e

e Propionate VeVProPropionateehhicpilcleioenate e VeVehhicilcle e

6 PZ-2891

6 PZ-2891 80 80 PZ-2891 6 PZ-2891

1 60 1 60

60 l

m m 40 m 40

(

40 ( 40

a 20

a 20 a d 20

A 0 0 A 0

CoA Acceetytyl Clo A Proopiponioyln CyolA CoACoACoA AAcAecctycelety tyCtylo lCA lo A ProopPripooniopoyipoln CniyoollAn CyolA CoACoA AAcectyel tyColA Proopiponioyln CyolA CoA CoA CoA CoA CoACoA CoACoA CoA CoA

)

C D 15 Vehicle E ) s 30 VehCicle 150 Vehicle D 15 Vehicle E s Vehicle 150

e 30 Vehicle l Vehicle

e Vehicle

l CPCA l CPCA

c Vehicle ) e Vehicle CPCA UK5099 c Vehicle ) i e Vehicle CPCA UK5099 UK5099 i UK5099

CPCA h

% CPCA CPCA %

(

6 CPCA

(

I 100

10 20 I 100

1 20 .

(

l e 5 50

l 10 e 5 50 c 10

b 0 b 0 0 A 0

A 0

CoA Acetyl CoA Propionyl CoA CPC CoA M0 M1 M2 CPC CoA M0 M1 M2 CoA Acetyl Propionyl CPC CoA CoACoA AAM0cectyel tyColA PrPropoiM1opniyol nCyoAl M2 CPC CoA M0 M1 M2 CoA CoA CoA CoA Figure 6. TheThe sample sample preparation preparation and and method method successfully successfully detects detects expected expected perturbations perturbations in CoA in metabolism: CoA metabolism: (A–D) (HepG2A–D) HepG2 cells werecells treated were treatedfor 24 h. for with 24 1 h. mM with propionate 1 mM propionate (A), 10 μM (A PZ), 10-2891µM (B PZ-2891), or 1 mM (B), cyclopropanecarboxylic or 1 mM cyclopropanecar- acid boxylic(CPCA;acid C,D (CPCA;). CoA andC,D short). CoA-chain and short-chainacyl CoA esters acyl CoAwere esters extracted were and extracted measured and measuredas described as described above. (E above.) Mass (isotopo-E) Mass 13 logue distribution (M.I.D.) of acetyl CoA extracted from HepG2 cells treated for 24 h. with U- C613-glucose. All data are isotopologue distribution (M.I.D.) of acetyl CoA extracted from HepG2 cells treated for 24 h. with U- C6-glucose. All data presented as mean  standard error of the mean (S.E.M.) for n = 3 technical replicates. are presented as mean ± standard error of the mean (S.E.M.) for n = 3 technical replicates.

Having demonstrated thatthat thethe methodmethod can can detect detect increases increases in in steady-state steady-state CoA CoA levels, lev- weels, thenwe then measured measured whether whether it canit can similarly similarly detect detect decreased decreased levels levels of of CoACoA andand CoA esters. We We treated treated HepG2 HepG2 cells cells with with cyclopropanecarboxylic cyclopropanecarboxylic (CPCA), (CPCA), which which is known is known to decreaseto decrease CoA CoA levels levels in mammalian in mammalian hepatocytes hepatocytes [35] [35. Indeed,]. Indeed, CPCA CPCA treatment treatment halved halved in- tracellularintracellular free free CoA CoA levels levels and and reduced reduced the thelevels levels of short of short-chain-chain acyl acyl CoAs CoAs including including ace- tylacetyl and and propionyl propionyl CoA CoA (Figure (Figure 6C).6C). As As expected, expected, cyclopropanecarboxyl cyclopropanecarboxyl-CoA-CoA levelslevels werewere increased (Figure6 6D),D),suggesting suggestingthat thatformation formationof ofthis this acyl-CoAacyl-CoA isis likelylikely associatedassociated withwith the depletion of fre freee CoA. Finally, we determined ifif our method could be used to measure incorporation of of isotopically isotopically labeled labeled substrates substrates into into short-chain short-chain acyl CoAs. acyl CoAs.Treating Treating HepG2 13 13 HepG2cells with cells uniformly with uniformly labelled labelledC6-glucose C6-glucose exhibited exhibited incorporation incorporation into acetyl into CoAacetyl (“M2” CoA (“M2”isotopologue) isotopologue) that was that substantially was substantially decreased decreased in response in response to UK5099 to UK5099 [36], a[36] potent, a potent and andspeciﬁc specific inhibitor inhibitor of mitochondrial of mitochondrial pyruvate pyruvate uptake uptake (Figure (Figure6E). 6E).

Metabolites 2021, 11, 468 12 of 18

2.8. The Method Detects Changes in the CoA Biosynthetic Pathway and Short-Chain Acyl CoA Species in IL-4-Polarized Macrophages Treated with Excess Etomoxir Finally, we used our method to follow-up previous studies demonstrating the macrophage response to the anti-inﬂammatory cytokine IL-4 is inhibited by high concentrations of etomoxir. Treating macrophages with 200 µM etomoxir depleted free CoA (as measured by colorimetric assay) and qualitatively increased levels of pantothenate Figure 7 (measured separately by LC-MS/MS) [12]. We previously speculated high concentrations of etomoxir may be sufﬁcient to deplete steady-state CoA levels by formation of an etomoxiryl CoA ester [12]. However, the ability to accurately quantify pantothenate, A 250 IL-4 C IL-4+dephospho-CoA, eto. and short-chain CoA esters in the same method allowed us to test the 200 cells) hypothesis that the etomoxiryl CoA ester may inhibit pantothenate kinase (PANK) similarly 6 150 to feedback inhibition by other long-chain CoA esters [37]. Consistent with previous work, 100 treatment with 200 µM etomoxir increased intracellular pantothenate levels (Figure7A) 50 and decreased steady-state free CoA levels (Figure7B). Additionally, our LC-MS/MS 0.3 method also revealed a reduction in dephospho-CoA (Figure7A), supporting a mechanism whereby etomoxir depletes free CoA in part by PANK inhibition (Figure7C). We also Figure 7 observed a reduction in other short-chain acyl CoAs in macrophages, notably acetyl CoA 0.1 (Figure7B), highlighting that the etomoxir-induced disruption of CoA homeostasis in A 250 macrophagesIL-4 perturbs multiple aspectsC of short-chain CoA biology. 0.0 IL-4+ eto. Abundance (pmol/1x10 200

cells) Pantothenate Dephospho-CoA

6 150 250 IL-4 30 A IL-4+ eto. IL-4 C B 200 Pantothenate(intracellular) 100 cells) IL-4+ eto. ATP 6 150 PANK cells)

6 ADP 50 100 20 0.3 50 4’-Phoshopantothenate ATP, Cysteine 0.3 PPCS AMP, PPi

0.1 0.1 4'-phosphopantothenoylcysteine 10 PPCDC 0.0 CO2 Abundance (pmol/1x10 0.0 Pantothenate Dephospho-CoA Abundance (pmol/1x10 Pantothenate Dephospho- Pantothenate Dephospho-CoACoA 4'-phosphopantetheine B 30 IL-4 ATP

Abundance (pmol/1x10 0 30 IL-4 IL-4+ eto. PPAT (COASY subunit) PPi

B CoA cells) Acetyl CoA Propionyl CoA 6 IL-4+ eto.

cells) 20 Dephospho-coenzyme A 6

ATP 20 DPCK (COASY subunit) ADP 10 Coenzyme A EtomoxirAcyl group

10 Abundance (pmol/1x10 0 CoACoA Acetyl CoA PropionylPropionyl CoA CoA CoA Etomoxiryl CoA

Figure 7. The SSA sample preparation and LC-MS/MS method detects changes in CoA synthesis

Abundance (pmol/1x10 0 CoApathway intermediatesAcetyl CoA andPropionyl short-chain CoA acyl CoA species in IL-4-polarized macrophages following etomoxir treatment. Data are presented as mean ± standard error of the mean (S.E.M.) for n = 3 technical replicates. 3. Discussion Here we detail an LC-MS/MS method that allows for the sensitive detection of short- chain acyl CoAs and metabolites in the CoA biosynthetic pathway. Our method efﬁciently overcomes limitations that otherwise preclude the use of a single method to analyze CoA esters and biosynthetic precursors. In particular, use of SSA circumvents the need for solid phase extraction (SPE) [17,31] as this single solvent can be used as both a deproteinizing agent and solvent for the reconstitution of acyl CoAs. There are multiple advantages gained from bypassing SPE with the use of SSA. SPE results in the poor recovery of free CoA and biosynthetic intermediates. Although the divergent polarities between the two classes of analytes can lead to poor chromatography, this can be corrected by carefully adjusting the pH, including an ion pairing agent, and Metabolites 2021, 11, 468 13 of 18

using an ultra-high performance liquid chromatography column. Eliminating SPE also circumvents the need to run multiple internal standards to correct for the differential recovery of various CoA esters. As many of these are not commercially available, intensive and specialized techniques—such as generation of SILEC-labeled internal standards—are often required [21,22]. The single, SSA extraction step, however, allows for the use of a single commercially available internal standard (crotonyl CoA). We demonstrate that endogenous levels of crotonyl CoA range from below 1% (cultured cells) to 3% (5 mg murine liver) of the internal standard, indicating that changes in endogenous crotonyl CoA levels would introduce minimal error when quantifying short-chain CoAs and CoA biosynthetic intermediates. However, caution should be exercised when interpreting data in model systems where steady-state crotonyl CoA levels are high or may substantially change between experimental groups, and SILEC-generated standards may be necessary. Proof-of-concept biological experiments demonstrate that the proposed method is sensitive and can be used to quantify CoA-related metabolism in biological samples. In addition to establishing that this method can be used to detect both augmentation and depletion of acyl CoAs following pharmacological perturbations, the simultaneous quantification of metabolites involved in CoA biosynthesis may have biological and clinical utility. For example, more than 50% of neurodegeneration with brain iron accumulation (NBIA) cases are caused by dysfunction of enzymes involved in CoA synthesis [9,38]. Patient fibroblasts and tissues from preclinical disease models are often characterized by decreased free CoA [39,40], though mutations in both pantothenate kinase (PANK) or coenzyme A synthase (COASY) can cause NBIA [9,38]. As such, a combined LC-MS/MS method detecting short-chain acyl CoAs as well as the reactants and products of enzymes involved in NBIAs may be useful in better identifying the molecular basis resulting in these pathologies. This method also proved useful in better understanding how etomoxir perturbs intracellular CoA homeostasis in IL-4-activated macrophages [12]. We were able to advance previous work by demonstrating that excess concentrations of etomoxir increased intracellular pantothenate levels while dephospho-CoA was decreased. This finding suggests that etomoxir may disrupt CoA metabolism by inhibiting a process downstream of pantothenate uptake but upstream of dephospho-CoA production. Given that long-chain acyl CoAs are potent inhibitors of PANK [37,41], it is likely that etomoxir is converted to its CoA thioester etomoxiryl CoA to inhibit PANK and block CoA biosynthesis. This reduction in steady-state free CoA in BMDMs is also associated with a reduction in short-chain acyl CoAs such as acetyl CoA, propionyl CoA, and succinyl CoA. As these metabolites are involved in many facets of cell physiology including the Krebs cycle, sterol synthesis, post-translational modifications, and epigenetic reprogramming, the mechanism linking disrupted CoA homeostasis by etomoxir with a reduced IL-4 response remains unclear.

4. Materials and Methods All animal procedures were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the UCLA Animal Research Committee (ARC) under protocol #2020-027-1.

4.1. Materials and Reagents DMEM (Cat#11965) was purchased from ThermoFisher (Waltham, MA, USA). Oasis HLB SPE columns were purchased from Waters (Milford, MA, USA). 5-sulfosalicylic acid, etomoxir, pantothenate, dephospho-CoA, and acyl CoA standards were purchased from Sigma-Aldrich (St. Louis, MO, USA). (E)-but-2-enoyl Coenzyme A (crotonoyl CoA) was purchased from Avanti Polar Lipids (Alabaster, Alabama, USA).

4.2. Cell Lines and Culture HepG2 cells were obtained from American Type Culture Collection (ATCC) and cultured according to ATCC recommendations. Cells were maintained in MEM with 10% (v/v) Metabolites 2021, 11, 468 14 of 18

FBS and 1% (v/v) penicillin/streptomycin. For biological validation experiments, 3.0 × 106 cells were seeded in 10 cm dishes. After 48 h., cells were treated with 1 mM sodium propionate (Sigma-Aldrich), 1 mM cyclopropanecarboxylic acid (Sigma-Aldrich), 10 µM PZ-2891 (MedChem Express), or 5 µM UK5099 (Sigma-Aldrich) for 24 h. and harvested for extractions. The 293FT cell line was obtained from Invitrogen and maintained in DMEM with 10% FBS and 1% penicillin/streptomycin. For acyl CoA extractions, 2.0 × 106 cells were seeded in 10 cm dishes and harvested after 72 h. Bone marrow-derived macrophages (BMDMs) were harvested by ﬂushing the tibiae and femurs of 8–12-week male mice. Fol- lowing red blood cell lysis, cells were centrifuged at 364× g for 5 min and resuspended in DMEM supplemented with 10% (v/v) FBS, 1% (v/v) penicillin/streptomycin, and a 5% (v/v) CMG-conditioned medium [42]. BMDMs were differentiated for 6 days and 5.0 × 106 BMDMs were then re-plated in 10 cm dishes. After 48 h., new medium containing 20 ng/mL IL-4 (Peprotech) alone or IL-4 with 200 µM etomoxir was added to cells for 24 h. before extraction.

4.2.1. Snap-Freezing of Cell Pellets Cells in 10 cm plates were washed with 5 mL of phosphate buffered saline (PBS) and harvested by scraping in 1 mL of PBS before being transferred to 1.5 mL microfuge tubes. The cells were centrifuged at 1500× g at 4 ◦C for 5 min. The supernatant was removed and cell pellets were snap-frozen by with liquid nitrogen.

4.2.2. Extraction of Pathway Intermediates and CoA Species from Cell Pellets with 5-Sulfosalicylic Acid (SSA) or Trichloroacetic Acid (TCA) For SSA extraction, a 2.5% (w/v) SSA solution with 1 µM crotonoyl CoA was prepared. An extraction solution of 200 µL was added to cell pellets on wet ice and mixed. The samples were then centrifuged at 18,000× g for 15 min. Following centrifugation, supernatants were removed and transferred to glass LC-MS vials for analysis. For extraction with 10% (w/v) TCA, a solution containing 1 µM crotonoyl CoA was prepared. An extraction solution of 200 µL was added to cell pellets and samples were resuspended by gentle pipetting. Solid phase extraction was then performed according to published methods [16,21,22]. Brieﬂy, Oasis HLB columns (Waters) were ﬁrst conditioned with 1 mL methanol, then equilibrated with 1 mL H2O. After equilibration, TCA-extracted samples were placed onto columns, washed with 1 mL H2O, and eluted with 1 mL of 25 mM ammonium acetate in methanol. Samples were dried overnight at 4 ◦C in a Centrivap benchtop vacuum concentrator (Labconco; St. Louis, MO, USA). Evaporated samples were reconstituted with 2.5% SSA and moved to LC-MS vials for analysis.

4.3. ESI LC-MS/MS Analytical Method 4.3.1. Liquid Chromatography Chromatography was performed on an Agilent 1290 ultra-high performance liquid chromatography (UHPLC) system using a Phenomenex Kinetex UHPLC C18 column (2.6 µm particle size, 2.1 mm ID × 150 mm). For analysis, 10 µL of sample was injected per run with a ﬂow rate of 300 µL/min. The chromatography settings are detailed in Table2.

4.3.2. Mass Spectrometry Mass spectrographic analysis was performed on a SCIEX 5500 QTrap run in both positive and negative modes and controlled by Analyst 1.6.2 software. Two separate MRM transitions were determined for each compound, and DP, EP, CE, and CXP for each compound were all manually optimized by tuning on direct infusions of approximately 100 ng compound per mL of 35/65 solvents A and B (see Figure2A and Supplemental Table S1 for ﬁnal settings). The source settings were optimized by tuning on a mix of analytes while also ﬂowing in 35/65 solvents A and B at 300 uL/min with temperature set at 450 ◦C; GS1 and GS2 at 45; curtain gas at 35; CAD gas at medium; ion spray voltage in positive mode = 5500 volts; and ion spray voltage in negative mode = –4500 volts. Metabolites 2021, 11, 468 15 of 18

4.3.3. Data Analysis Peaks were integrated and concentrations were calculated using MultiQuant software (SCIEX). The concentration of each analyte was determined from the ratio of the analyte to its internal standard by a calibration curve. Curves were generated in an extraction solution and a linear regression with 1/x weighting was performed for each curve to determine the line of best ﬁt. Data for biological samples was normalized to cell number.

4.4. Validation of Sample Preparation and LC-MS/MS Methods Validation was performed in accordance with both the FDA guidelines for bioanalytical method validation [32] and the European Medicines Agency guidelines on bioanalytical method validation [33,43].

4.4.1. Linearity, Lower Limit of Detection (LLOD), and Lower Limit of Quantitation (LLOQ) Calibration standards were spiked into the extraction solution across a wide range of concentrations involving at least 6–8 concentrations, and an internal standard was added to all calibrant solutions at the concentration matching biological samples. The allowable bias was within 15% for all calibrations for ≥75% of the standards except at the LLOQ, for which the allowable bias was within 20%. Linearity was determined by the correlation coefﬁcient of the linear regression with 1/x weighting. LLOQ was determined as the lowest value in the calibration range for which bias was within 20%. LLOD was determined as the lowest value in the concentration range for which the peak height of the analyte chromatogram was ﬁve-fold greater than the baseline signal.

4.4.2. Matrix Effect and Comparative Accuracy Approximately 2.0 × 107 293FT cells were divided into 20 equal pellets that were extracted in the SSA as above. Standards were then spiked into groups of 5 post-extraction matrix samples at 0 (0 pmol), low (62.5 pmol), medium (250 pmol) and high (1000 pmol) levels. Low, medium, and high standards were also spiked into equal volumes of the extraction solvent (n = 5 per group) and thus there were 7 separate groups of 5 replicates each. The group average of the AUC for each analyte in each group was determined. To determine the matrix effect, the baseline mean AUC for each analyte in post-extraction matrix (0 pmol spike group) was ﬁrst subtracted from the mean AUC for each analyte in the low, medium, and high spikes into post-extraction matrix. These baseline-subtracted means were then compared to the mean AUC for each analyte in the low, medium, and high spikes in the extraction solvent. The matrix effect was reported as the percentage-based ratio of this comparison. Following the determination of the matrix effect upon AUC, analyte concentrations were determined for all groups. Comparative accuracy was determined as the percentage-based ratio of the mean baseline-subtracted concentrations for the spikes in the post-extraction matrix relative to the mean concentrations for those spikes in the extraction solution. In general, the internal standard corrected matrix effect was deemed acceptable if it produced comparative accuracy of within 15%.

4.4.3. Precision and Accuracy Approximately 2.0 × 107 293FT cells were divided into 20 equal pellets that were extracted in the SSA as above. Low (62.5 pmol), medium (250 pmol), or high (1000 pmol) amounts of all eight core standards in addition to controls lacking standards were spiked into groups of 5 pellets. The SSA extraction solution was added to all 20 samples and the concentration was calculated for each analyte for each sample. Precision was determined for each analyte across the 5 replicates at each concentration (0, 62.5, 250, and 1000 pmol) as the relative standard deviation or coefﬁcient of variation (CV). In general, precision was deemed acceptable with CV ≤ 15%. Accuracy was determined by comparing the average calculated concentration for each analyte at each spiked concentration to the nominal value, which was the amount spiked at that concentration added to the value determined for the Metabolites 2021, 11, 468 16 of 18

baseline or 0 group. In general, accuracy was deemed acceptable when mean values were within 15% of the nominal values.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/metabo11080468/s1, Figure S1. The CoA biosynthetic pathway with corresponding structures, Figure S2. Pantothenate is only detectable with adequate sensitivity with negativemode MRM, Figure S3. Free CoA is produced in the ESI source from acetyl, propionyl, and isovaleryl CoA, Figure S4. Endogenous crotonoyl CoA concentrations, Table S1. Mass spectrometry parameters for detection of short-chain acyl CoAs, dephospho-CoA, and pantothenate, Table S2. Determination of matrix effect. Author Contributions: A.E.J., A.S.D., S.T.R. and D.M. conceived the study and planned the experiments. A.E.J., N.J.A., A.A. and D.M. conducted the experiments. A.E.J., N.J.A., A.S.D. and D.M. prepared the figures. A.E.J., N.J.A., A.S.D. and D.M. wrote the initial draft of the manuscript with all authors approving the final draft. All authors have read and agreed to the published version of the manuscript. Funding: This work was generously supported by the National Institute of Health (R35GM138003, P30DK063491, and P50CA092131 to A.S.D.). A.E.J. was supported by the UCLA Tumor Cell Biology Training Grant (T32CA009056). This study was further supported in part by US Public Health Service Research Grants R01 HL129051 (S.T.R.) and the Laubisch, Castera, and M.K. Grey Funds of the University of California at Los Angeles. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data is available from the corresponding authors upon request. Acknowledgments: We are grateful to Krista Yang and Andréa B. Ball for their assistance with cell culture, acyl CoA extractions, and manuscript review. Conflicts of Interest: The authors declare no conflict of interest.

References 1. Leonardi, R.; Zhang, Y.-M.; Rock, C.O.; Jackowski, S. Coenzyme A: Back in action. Prog. Lipid Res. 2005, 44, 125–153. [CrossRef] 2. Pietrocola, F.; Galluzzi, L.; Pedro, J.M.B.-S.; Madeo, F.; Kroemer, G. Acetyl Coenzyme A: A Central Metabolite and Second Messenger. Cell Metab. 2015, 21, 805–821. [CrossRef][PubMed] 3. Prasad, P.D.; Wang, H.; Kekuda, R.; Fujita, T.; Fei, Y.-J.; Devoe, L.D.; Leibach, F.H.; Ganapathy, V. Cloning and Functional Expression of a cDNA Encoding a Mammalian Sodium-dependent Vitamin Transporter Mediating the Uptake of Pantothenate, Biotin, and Lipoate. J. Biol. Chem. 1998, 273, 7501–7506. [CrossRef][PubMed] 4. Quick, M.; Shi, L. The Sodium/Multivitamin Transporter: A multipotent system with therapeutic implications. Vitam. Horm. 2015, 98, 63–100. [CrossRef][PubMed] 5. Robishaw, J.D.; Neely, J.R. Pantothenate kinase and control of CoA synthesis in heart. Am. J. Physiol. 1984, 246, H532–H541. [CrossRef][PubMed] 6. Rivera, L.G.; Bartlett, M.G. Chromatographic methods for the determination of acyl-CoAs. Anal. Methods 2018, 10, 5252–5264. [CrossRef] 7. Vessey, D.A.; Kelley, M.; Warren, R.S. Characterization of the CoA ligases of human liver mitochondria catalyzing the activation of short- and medium-chain fatty acids and xenobiotic carboxylic acids. Biochim. Biophys. Acta 1999, 1428, 455–462. [CrossRef] 8. Gauthier, N.; Wu, J.W.; Wang, S.P.; Allard, P.; Mamer, O.A.; Sweetman, L.; Moser, A.B.; Kratz, L.; Álvarez, F.; Robitaille, Y.; et al. A Liver-Speciﬁc Defect of Acyl-CoA Degradation Produces Hyperammonemia, Hypoglycemia and a Distinct Hepatic Acyl-CoA Pattern. PLoS ONE 2013, 8, e60581. [CrossRef] 9. Venco, P.; Dusi, S.; Valletta, L.; Tiranti, V. Alteration of the coenzyme A biosynthetic pathway in neurodegeneration with brain iron accumulation syndromes. Biochem. Soc. Trans. 2014, 42, 1069–1074. [CrossRef] 10. Gregory, A.; Hayﬂick, S.J. Pantothenate Kinase-Associated Neurodegeneration. In GeneReviews®; Adam, M.P., Ardinger, H.H., Pagon, R.A., Wallace, S.E., Bean, L.J.H., Mirzaa, G., Amemiya, A., Eds.; University of Washington: Seattle, WA, USA, 1993. 11. Covarrubias, A.; Aksoylar, H.I.; Yu, J.; Snyder, N.; Worth, A.J.; Iyer, S.S.; Wang, J.; Ben-Sahra, I.; Byles, V.; Polynne-Stapornkul, T.; et al. Akt-mTORC1 signaling regulates Acly to integrate metabolic input to control of macrophage activation. eLife 2016, 5, e11612. [CrossRef] 12. Divakaruni, A.S.; Hsieh, W.Y.; Minarrieta, L.; Duong, T.N.; Kim, K.K.; Desousa, B.R.; Andreyev, A.Y.; Bowman, C.E.; Caradonna, K.; Dranka, B.P.; et al. Etomoxir Inhibits Macrophage Polarization by Disrupting CoA Homeostasis. Cell Metab. 2018, 28, 490–503.e7. [CrossRef][PubMed] Metabolites 2021, 11, 468 17 of 18

13. Neubauer, S.; Chu, D.B.; Marx, H.; Sauer, M.; Hann, S.; Koellensperger, G. LC-MS/MS-based analysis of coenzyme A and short-chain acyl-coenzyme A thioesters. Anal. Bioanal. Chem. 2015, 407, 6681–6688. [CrossRef][PubMed] 14. Tsuchiya, Y.; Pham, U.; Gout, I. Methods for measuring CoA and CoA derivatives in biological samples. Biochem. Soc. Trans. 2014, 42, 1107–1111. [CrossRef][PubMed] 15. Perera, M.A.D.; Choi, S.-Y.; Wurtele, E.; Nikolau, B.J. Quantitative analysis of short-chain acyl-coenzymeAs in plant tissues by LC–MS–MS electrospray ionization method. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 2009, 877, 482–488. [CrossRef] [PubMed] 16. Snyder, N.; Basu, S.S.; Worth, A.J.; Mesaros, C.; Blair, I.A. Metabolism of propionic acid to a novel acyl-coenzyme A thioester by mammalian cell lines and platelets. J. Lipid Res. 2015, 56, 142–150. [CrossRef] 17. Gao, L.; Chiou, W.; Tang, H.; Cheng, X.; Camp, H.S.; Burns, D.J. Simultaneous quantification of malonyl-CoA and several other short-chain acyl-CoAs in animal tissues by ion-pairing reversed-phase HPLC/MS. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 2007, 853, 303–313. [CrossRef] 18. Park, J.W.; Jung, W.S.; Park, S.R.; Park, B.C.; Yoon, Y.J. Analysis of intracellular short organic acid-coenzyme A esters from actinomycetes using liquid chromatography-electrospray ionization-mass spectrometry. J. Mass Spectrom. 2007, 42, 1136–1147. [CrossRef] 19. Hayashi, O.; Satoh, K. Determination of Acetyl-CoA and Malonyl-CoA in Germinating Rice Seeds Using the LC-MS/MS Technique. Biosci. Biotechnol. Biochem. 2006, 70, 2676–2681. [CrossRef][PubMed] 20. Minkler, P.E.; Kerner, J.; Ingalls, S.T.; Hoppel, C.L. Novel isolation procedure for short-, medium-, and long-chain acyl-coenzyme A esters from tissue. Anal. Biochem. 2008, 376, 275–276. [CrossRef] 21. Basu, S.S.; Blair, I.A. SILEC: A protocol for generating and using isotopically labeled coenzyme A mass spectrometry standards. Nat. Protoc. 2011, 7, 1–11. [CrossRef] 22. Basu, S.S.; Mesaros, C.; Gelhaus, S.L.; Blair, I.A. Stable Isotope Labeling by Essential Nutrients in Cell Culture for Preparation of Labeled Coenzyme A and Its Thioesters. Anal. Chem. 2011, 83, 1363–1369. [CrossRef][PubMed] 23. Sedgwick, G.W.; Fenton, T.W.; Thompson, J.R. Effect of protein precipitating agents on the recovery of plasma free amino acids. Can. J. Anim. Sci. 1991, 71, 953–957. [CrossRef] 24. Berrueta, L.A.; Gallo, B.; Vicente, F. A review of solid phase extraction: Basic principles and new developments. Chromatographia 1995, 40, 474–483. [CrossRef] 25. Abrankó, L.; Williamson, G.; Gardner, S.; Kerimi, A. Comprehensive quantitative analysis of fatty-acyl-Coenzyme A species in biological samples by ultra-high performance liquid chromatography–tandem mass spectrometry harmonizing hydrophilic interaction and reversed phase chromatography. J. Chromatogr. A 2018, 1534, 111–122. [CrossRef][PubMed] 26. Palladino, A.A.; Chen, J.; Kallish, S.; Stanley, C.; Bennett, M.J. Measurement of tissue acyl-CoAs using flow-injection tandem mass spectrometry: Acyl-CoA profiles in short-chain fatty acid oxidation defects. Mol. Genet. Metab. 2012, 107, 679–683. [CrossRef] 27. Chemdata: Coenzyme A. Available online: https://www.drugfuture.com/chemdata/coenzyme-a.html (accessed on 25 April 2021). 28. Pubchem Compound Summary: N,N-dimethybutylamine. Available online: https://pubchem.ncbi.nlm.nih.gov/compound/N_ N-Dimethylbutylamine (accessed on 25 April 2021). 29. Basiri, B.; Murph, M.M.; Bartlett, M.G. Assessing the Interplay between the Physicochemical Parameters of Ion-Pairing Reagents and the Analyte Sequence on the Electrospray Desorption Process for Oligonucleotides. J. Am. Soc. Mass Spectrom. 2017, 28, 1647–1656. [CrossRef] 30. Demoz, A.; Garras, A.; Asiedu, D.K.; Netteland, B.; Berge, R.K. Rapid method for the separation and detection of tissue short-chain coenzyme A esters by reversed-phase high-performance liquid chromatography. J. Chromatogr. B Biomed. Sci. Appl. 1995, 667, 148–152. [CrossRef] 31. Khan, K.; Blaak, E.; Elia, M. Quantifying intermediary metabolites in whole blood after a simple deproteinization step with sulfosalicylic acid. Clin. Chem. 1991, 37, 728–733. [CrossRef] 32. U.S. Department of Health and Human Services: Food and Drug Administration Center for Drug Evaluation and Research and Center for Veterinary Medicine. Guidance for Industry, Bioanalytical Method Validation. 2001. Available online: http: //www.fda.gov/cder/guidance/index.htm (accessed on 25 April 2021). 33. Smith, G. European Medicines Agency guideline on bioanalytical method validation: What more is there to say? Bioanalysis 2012, 4, 865–868. [CrossRef] 34. Sharma, L.K.; Subramanian, C.; Yun, M.-K.; Frank, M.W.; White, S.W.; Rock, C.O.; Lee, R.E.; Jackowski, S. A therapeutic approach to pantothenate kinase associated neurodegeneration. Nat. Commun. 2018, 9, 4399. [CrossRef] 35. Ulrich, R.G.; Bacon, J.A.; Brass, E.P.; Cramer, C.T.; Petrella, D.K.; Sun, E.L. Metabolic, idiosyncratic toxicity of drugs: Overview of the hepatic toxicity induced by the anxiolytic, panadiplon. Chem. Biol. Interact. 2001, 134, 251–270. [CrossRef] 36. Divakaruni, A.S.; Wallace, M.; Buren, C.; Martyniuk, K.; Andreyev, A.Y.; Li, E.; Fields, J.A.; Cordes, T.; Reynolds, I.J.; Bloodgood, B.; et al. Inhibition of the mitochondrial pyruvate carrier protects from excitotoxic neuronal death. J. Cell Biol. 2017, 216, 1091–1105. [CrossRef] 37. Rock, C.O.; Karim, M.A.; Zhang, Y.-M.; Jackowski, S. The murine pantothenate kinase (Pank1) gene encodes two differentially regulated pantothenate kinase isozymes. Gene 2002, 291, 35–43. [CrossRef] Metabolites 2021, 11, 468 18 of 18

38. Di Meo, I.; Carecchio, M.; Tiranti, V. Inborn errors of coenzyme a metabolism and neurodegeneration. J. Inherit. Metab. Dis. 2019, 42, 49–56. [CrossRef][PubMed] 39. Dusi, S.; Valletta, L.; Haack, T.B.; Tsuchiya, Y.; Venco, P.; Pasqualato, S.; Goffrini, P.; Tigano, M.; Demchenko, N.; Wieland, T.; et al. Exome Sequence Reveals Mutations in CoA Synthase as a Cause of Neurodegeneration with Brain Iron Accumulation. Am. J. Hum. Genet. 2014, 94, 11–22. [CrossRef][PubMed] 40. Garcia, M.; Leonardi, R.; Zhang, Y.-M.; Rehg, J.E.; Jackowski, S. Germline Deletion of Pantothenate Kinases 1 and 2 Reveals the Key Roles for CoA in Postnatal Metabolism. PLoS ONE 2012, 7, e40871. [CrossRef][PubMed] 41. Rock, C.O.; Calder, R.B.; Karim, M.A.; Jackowski, S. Pantothenate Kinase Regulation of the Intracellular Concentration of Coenzyme A. J. Biol. Chem. 2000, 275, 1377–1383. [CrossRef][PubMed] 42. Hsieh, W.-Y.; Williams, K.J.; Su, B.; Bensinger, S.J. Proﬁling of mouse macrophage lipidome using direct infusion shotgun mass spectrometry. STAR Protoc. 2021, 2, 100235. [CrossRef][PubMed] 43. Lynch, K.L. CLSI C62-A: A New Standard for Clinical Mass Spectrometry. Clin. Chem. 2016, 62, 24–29. [CrossRef][PubMed]