Development of Tandem Mass Tag Labeling Method for Lipid Molecules Containing Carboxy and Phosphate Groups, and Their Stability in Human Serum

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Article Development of Tandem Mass Tag Labeling Method for Lipid Molecules Containing Carboxy and Phosphate Groups, and Their Stability in Human Serum

Suzumi M. Tokuoka 1, Yoshihiro Kita 1, Masaya Sato 2 , Takao Shimizu 1,3, Yutaka Yatomi 2 and Yoshiya Oda 1,*

1 Department of Lipidomics, Graduate School of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-8654, Japan; [email protected] (S.M.T.); [email protected] (Y.K.); [email protected] (T.S.) 2 Department of Clinical Laboratory Medicine, Graduate School of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-8654, Japan; [email protected] (M.S.); [email protected] (Y.Y.) 3 Department of Lipid Signaling, National Center for Global Health and Medicine, Toyama 1-21-1, Shinjuku-ku, Tokyo 162-8655, Japan * Correspondence: [email protected]; Tel.: +81-3-5841-3540

Abstract: In clinical lipidomics, it is a challenge to measure a large number of samples and to reproduce the quantitative results. We expanded the range of application of the tandem mass tag (TMT) method, which is widely used in proteomics, to lipidomic fields. There are various types of lipid molecule, for example, eicosanoids have a carboxyl group and phosphatidic acid has a phosphate group. We modified these functional groups simultaneously with TMT. This approach allows for a single analysis by mixing six samples and using one of the six samples as a bridging sample; the quantitative data can be easily normalized even if the number of measurements increases. To accommodate a large number of samples, we utilize a pooled serum sample of 300 individuals as Citation: Tokuoka, S.M.; Kita, Y.; Sato, a bridging sample. The stability of these lipid molecules in serum was examined as an analytical M.; Shimizu, T.; Yatomi, Y.; Oda, Y. validation for the simultaneous TMT labeling. It was found that the stability of these lipid molecules Development of Tandem Mass Tag in serum differs greatly depending on the lipid species. These findings reaffirmed the importance of Labeling Method for Lipid Molecules proper sample preparation and storage to obtain reliable data. The TMT labeling method is expected Containing Carboxy and Phosphate to be a useful method for lipidomics with high-throughput and reliable reproducibility. Groups, and Their Stability in Human Serum. Metabolites 2021, 11, 19. Keywords: high-throughput; relative quantitation; lipids with carboxy groups; lipids with phos- https://doi.org/10.3390/ phate groups metabo11010019

Received: 30 October 2020 Accepted: 28 December 2020 Published: 30 December 2020 1. Introduction Lipid molecules are involved in many physiological processes and are thought to Publisher’s Note: MDPI stays neu- be associated with a variety of diseases [1–3]. Omega-3 polyunsaturated fatty acids (PU- tral with regard to jurisdictional clai- FAs), including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), might be ms in published maps and institutio- beneficial for human health [4–6]. Phosphatidic acid (PA) is the simplest glycerophospho- nal affiliations. lipid present in cells. Recently, PA has also been thought to be a second messenger in signal transduction [7–9]; it is converted to diacylglycerol (DAG) by phosphohydrolases and is responsible for protein kinase C activation. In addition, PA is rapidly converted to lysophosphatidic acid (LPA) [10], a bioactive phospholipid present in various tissues, Copyright: © 2020 by the authors. Li- censee MDPI, Basel, Switzerland. by phospholipases. Quantitative analysis of these lipids in clinical samples is crucial for This article is an open access article understanding the function of bioactive lipids in physiology and various diseases. The distributed under the terms and con- most commonly used assay methods are gas chromatography (GC) or high-performance ditions of the Creative Commons At- liquid chromatography (HPLC) coupled to mass spectrometry (MS). The development of tribution (CC BY) license (https:// LC/MS lipidomic profiling has enabled the more comprehensive analysis of various types creativecommons.org/licenses/by/ of lipid molecule, and the importance of internal standards (ISs) was recognized soon after 4.0/). the advent of quantitative analysis by MS. In particular, the use of stable isotope-labeled

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lipids as ISs has evolved with the development of MS. However, it is not practical to prepare many stable isotope-labeled lipids for each target molecule. Furthermore, it is not easy to prepare the amount of ISs that need to be added to the sample in accordance with the concentration of the target molecule [11]. To solve this problem, Oda et al. developed a metabolic labeling method using stable isotope elements based on proteomics [12]. With this technique, accurate relative quantification was achieved without ISs; however, this method is limited to cells that can be cultured and is extremely difficult to apply to clinical specimens. Isotope-coded affinity tags [13] and isotope-coded protein labels [14] have been developed for this purpose. Although the relative quantification of proteins with reasonable accuracy became available with these methodologies, these methods require double target MS for measurements because of the isotope used for labeling. In addition, when relatively inexpensive deuterium is used for labeling, the target molecule is sometimes separated from the IS by HPLC or capillary GC, which results in differences in the ionization efficiency (ionization suppression) of the MS and thus causes quantitative discrepancies [15,16]. Therefore, methods such as Isobaric Tag for Relative and Absolute Quantitation [17] and tandem mass tag (TMT) [18] have been developed to quantify the target molecule using MS/MS product ions. In these methods, the precursor ion in MS is a common m/z, but the product ion m/z is different between the compared samples and, thus, the relative quantitation is performed by MS/MS. As the tags were developed for multiplexing, the number of measurements can be reduced because several compared samples are mixed together for measurement, which allows sample throughput to be increased. In the sixplex TMT method, six samples are measured in a single run. This multiplex method has advantages in not only throughput, but also relative quantification. Each TMT- derivatized molecule in the six samples elutes at exactly the same time, allowing the matrix effect among these six samples is exactly the same. Many papers in the field of proteomics have demonstrated the good reliability of relative quantification. Isobaric tag approaches in lipidomics [19] and metabolomics [20] for the compounds with amino groups have been used, the TMT labeling method for carboxy groups has been developed as a functional group other than amino groups [21], and we have previously conducted the quantitative analysis of free fatty acids using approximately 200 human plasma samples [22]. To take advantage of relative quantification by TMT labelling, we propose a strategy for using a common quality control (QC) sample each time for one of six sample in sixplex measurement, thereby allowing for comparisons of large number of sample sets as well as different datasets. In the present study, we developed a method for the simultaneous quantitative determination for carboxy-containing lipids, not only free fatty acids, but also their related metabolites, such as eicosanoids, as well as phosphate groups containing lipids, PAs, and looked at their stability in serum samples.

2. Results and Discussion 2.1. Lipid Extraction Methanol extraction method is simple and has a good recovery rates for medium- polarity lipids but do not remove many impurities that compete with chemical modification reactions (see below). In the blood, there are many polar metabolites with carboxyl and phosphate groups, such as organic acids, nucleic acids and amino acids. In the reaction, these molecules would compete with our TMT derivatization for lipids; therefore, the removal of such molecules by some lipid extraction methods is desirable. The extraction efficiency of LPA and hydroxylated PUFA, which have relatively high polarity, is poor in the Bligh–Dyer extraction method [23,24]. For practical reasons, we decided to perform 1-butanol extraction with the target organic layer on top and water layer at the bottom. Such lipid extraction methods with 1-buthanol have been used for a long time. Changing the sample pH is used to increase the extraction efficiency [23]; however, some lipids are unstable under both acidic and weakly alkaline conditions. For example, lysophosphatidylcholine (LPC), which exists in biological fluids, is degraded to LPA by acid, leading to an overestimation of LPA [25,26]. Considering the buffering capacity of the serum, we added Metabolites 2021, 11, x FOR PEER REVIEW 3 of 15

long time. Changing the sample pH is used to increase the extraction efficiency [23]; however, some lipids are unstable under both acidic and weakly alkaline conditions. For example, lysophosphatidylcholine (LPC), which exists in biological fluids, is degraded to Metabolites 2021, 11, 19 LPA by acid, leading to an overestimation of LPA [25,26]. Considering the buffering3 of 15 capacity of the serum, we added 1 M HCl to the serum at a final concentration of 0.1 M HCl and confirmed that the pH of the serum was 3 to 4. Then, the extraction was performed with1 M HCl1-buthanol. to the serum As shown at a final in concentration Figure 1A, the of 0.1 yield M HCl of lyso-phosphatidic and confirmed that acid the pH (18:1) of (LPA (18:1))the serum was was sufficient 3 to 4. Then, under the acidic extraction conditions; was performed however, with when 1-buthanol. we tested As shown the sample in of mixturesFigure1A, of the diacylphosphatidic yield of lyso-phosphatidic acid (18:1/18:1) acid (18:1) (LPA (PA (18:1))(36:2) wasand sufficient LPA (18:1), under approximately acidic twiceconditions; as much however, LPA was when recovered we tested thecompared sample ofto mixturesthe input, of and diacylphosphatidic the recovery of acid PA (36:2) was(18:1/18:1) inadequate. (PA (36:2) This and might LPA have (18:1), been approximately due to the twicefact that as much LPA LPA (18:1) was was recovered observed as a resultcompared of the to thedegradation input, and product the recovery of PA of PA(36:2) (36:2) in wasthe inadequate.starting material. This might To improve have the been due to the fact that LPA (18:1) was observed as a result of the degradation product of extraction efficiency, sodium chloride was added to the sample instead of using acidic PA (36:2) in the starting material. To improve the extraction efficiency, sodium chloride was conditions.added to the As sample shown instead in Figure of using 1, acidic an incr conditions.ease in AsLPA shown due into Figure degraded1, an increase PA was in not observed,LPA due and to degraded the yield PA was was sufficient. not observed, We andalso the confirmed yield was sufficient.that arachidonic We also confirmedacid and several eicosanoidsthat arachidonic also acidshowed and several good recovery eicosanoids rates also with showed this good butanol recovery extraction rates with method this using sodiumbutanol chloride extraction (Figure method 1B). using sodium chloride (Figure1B).

FigureFigure 1. 1.ExaminationExamination of of 1-buthanol extraction extraction conditions. conditions. The YThe-axis Y is-axis the ratiois the of ratio the peak of the area peak after extractionarea after to extraction the peak to the peakarea area without without the extraction the extracti process.on process. Error bars Error represent bars standardrepresent deviations. standard (A deviations.) Extraction efﬁciencies(A) Extraction of phosphatidic efficiencies acid of phosphatidic(PA) and acid lysophosphatidic (PA) and lysophosphatidic acid (LPA). The acid left and(LPA). middle The panels left and represent middle the panels recovery represent rate for 1-butanolthe recovery extraction rate for with 1-butanol extraction with each acidic or high salt condition. Mixture samples of 44 ng of LPA (18:1) and 50 ng of PA 18:1/18:1 (PA each acidic or high salt condition. Mixture samples of 44 ng of LPA (18:1) and 50 ng of PA 18:1/18:1 (PA (36:2) in a methanol (36:2) in a methanol stock solution were put in test tubes. After evaporation, 330 μL of phosphate buffered saline (PBS) stock solution were put in test tubes. After evaporation, 330 µL of phosphate buffered saline (PBS) was added, followed by was added, followed by 1-buthanol extraction with various conditions (acidic or salt). Three replicates were tested for 1-buthanol extraction with various conditions (acidic or salt). Three replicates were tested for extraction. (B) Extraction extraction. (B) Extraction efficiencies of several eicosanoids. The recovery of eicosanoids and arachidonic acid (AA) by efﬁciencies of several eicosanoids. The recovery of eicosanoids and arachidonic acid (AA) by 1-butanol extraction with each 1-butanol extraction with each salt condition is shown. A mixture of 5.1 ng of each 12-hydroxyeicosatetraenoic acid salt condition is shown. A mixture of 5.1 ng of each 12-hydroxyeicosatetraenoic acid (12-HETE), 20-hydroxyeicosatetraenoic (12-HETE), 20-hydroxyeicosatetraenoic acid (20-HETE), prostaglandin E2 (PGE2), thromboxane B2 (TXB2), and AA were acid (20-HETE), prostaglandin E (PGE ), thromboxane B (TXB ), and AA were put in the test tube. After evaporation, put in the test tube. After evaporation,2 2 30 μL of PBS was2 added2 followed by 1-buthanol extraction with the indicated 30 µL of PBS was added followed by 1-buthanol extraction with the indicated conditions. Three replicates were tested for conditions. Three replicates were tested for the extraction. the extraction. 2.2. Tandem Mass Tag (TMT) Derivatization 2.2. Tandem Mass Tag (TMT) Derivatization 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) is an easy-to-use and rela- tively1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide inexpensive cross-linker that is soluble in water (EDC) and is methanol, an easy-to-use forming and rela-active ester tively inexpensive cross-linker that is soluble in water and methanol, forming active ester intermediates with phosphate and carboxyl groups. It then undergoes nucleophilic sub- intermediates with phosphate and carboxyl groups. It then undergoes nucleophilic substi- stitutiontution in thein presencethe presence of strong of nucleophiles, strong nucleophiles, such as primary such amines. as EDCprimary cross-linking amines. EDC cross-linkingis commonly usedis commonly for the bioconjugation used for the of biocon DNA andjugation nucleic of acids DNA or and for bindingnucleic DNAacids or for bindingto microplate DNA wells to microplate under neutral wells conditions under neutral as one-pot conditions reaction methods as one-pot [27]. reaction For liquid- methods [27].phase For coupling, liquid-phase the reaction coupling, with the EDC reaction is almost with complete EDC is almost (95%) within complete 16 h (95%) at room within 16 htemperature at room temperature [28]. When [28]. we used When the we EDC used reaction the EDC to label reaction PAs with to aminoxyTMT,label PAs with the aminox- yTMT,reaction the proceeded reaction withoutproceeded any without problems. any Since problems. this reaction Since alsothis proceededreaction also for proceeded the forcarboxy the carboxy group, group, unreacted unreacted free fatty free acids fatty were acids observed were observed when the fattywhen acids the fatty and PAacids and reacted simultaneously. When EDC was added to the one-pot reaction solution containing PA reacted simultaneously. When EDC was added to the one-pot reaction solution con- 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) for the tainingTMT labeling 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium of free fatty acids, which we have already reported [22], no unreactedchloride (DMTMM) PAs or fatty acids were detected, and the progress of TMT formation based on PAs and free

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for the TMT labeling of free fatty acids, which we have already reported [22], no unre- Metabolites 2021, 11, 19 actedfor the PAs TMT or fatty labeling acids of were free detected,fatty acids, and which the progress we have of already TMT formation reported based [22], noon 4 unre-PAs of 15 andacted free PAs fatty or fattyacids acids was observed.were detected, When and there the wasprogress no EDC of TMT reagent formation and only based DMTMM on PAs wasand used, free fatty the TMTacids derivatizationwas observed. reaction When there proceeded was no to EDC free reagentfatty acids, and butonly it DMTMM did not reactfattywas acidswithused, PA. wasthe Figure observed.TMT derivatization 2 shows When these there reaction chemical was no pr EDCreactionoceeded reagent schemes to free and fattyonlyfor phosphor-lipids DMTMMacids, but wasit did used, and not carboxy-lipids.thereact TMT with derivatization PA. SinceFigure the 2 reaction shows modi ficationthese proceeded chemical of tophosphate free reaction fatty groups acids, schemes but by for it EDC did phosphor-lipids not cross-linking react with PA.and is widelyFigurecarboxy-lipids.2 used, shows it may these Since be chemical thepossible modi reaction tofication label schemes variousof phosphate phosphate for phosphor-lipids groups groups by withEDC and TMT. carboxy-lipids.cross-linking However, is phosphatidylcholine,Sincewidely the used, modification it may bephosphatidylethanolamine, of possible phosphate to label groups various by EDC phosphate and cross-linking their lyso-formsgroups is with widely could TMT. used, not However, itbe may la- beledbephosphatidylcholine, possible with TMT, to label probably various phosphatidylethanolamine, be phosphatecause two groups of the oxygen with and TMT. atoms their However, oflyso-forms the phosphorus phosphatidylcholine, could notatom be are la- bondedphosphatidylethanolamine,beled with to carbons. TMT, probably Input standard be andcause their compounds two lyso-forms of the withoutoxygen could atomsderivatization not be of labeledthe phosphorus was with not TMT, detectable atom prob- are indicatingablybonded because to derivatize carbons. two of Input theefficiency oxygen standard was atoms compounds sufficie of thent phosphorus (Figurewithout 3A). derivatization atom Representative are bonded was not spectra to detectable carbons. ob- tainedInputindicating standardby TMT derivatize reporter compounds efficiency ion-specific without was derivatizationprecursor sufficie ntion (Figure scanning was not 3A). detectable analysis Representative and indicating product spectra derivatize ion scan ob- spectraefficiencytained ofby derivatized wasTMT sufficient reporter PA, ion-specific (Figure Lyso-PA(LPA),3A). precursor Representative arachidonic ion scanning spectra acid and analysis obtained eicosanoid and by product TMT standards reporter ion scanare shownion-specificspectra in of Figure derivatized precursor 3B,C. ionPA, scanning Lyso-PA(LPA), analysis arachidonic and product acid ion and scan eicosanoid spectra of standards derivatized are PA,shown Lyso-PA(LPA), in Figure 3B,C. arachidonic acid and eicosanoid standards are shown in Figure3B,C.

Figure 2. ChemicalChemical reaction schemes. ( (AA)) Tandem Tandem mass mass tag tag ( (TMT)TMT) derivatization for the phosphate group. ( (BB)) TMT derivatizationFigure 2. Chemical for the reactioncarboxy group. schemes. (A) Tandem mass tag (TMT) derivatization for the phosphate group. (B) TMT derivatization for the carboxy group.

Figure 3. Cont.

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Figure 3. Derivatization of eicosanoids, fatty acids, lysophosphatidic acid and diacylphosphatidic acid. (A) Examples of Figure 3. Derivatization of eicosanoids, fatty acids, lysophosphatidic acid and diacylphosphatidic acid. (A) Examples of chromatograms for input standards before (upper) and after (lower) derivatization with se serum.rum. Input signal compounds without derivatization was almost undetectable after derivatizat derivatization.ion. Selective reaction reaction monito monitoringring (SRM) transitions were shown atat thethe toptop in in each each chromatogram chromatogram as as Q1 Q1 > Q3 > Q3 (polarity). (polarity). Injection Injection volume volume was was 5 µL 5 and μL theand concentration the concentration of compounds of com- poundswere 0.85 were ng/ µ0.85L each ng/μ forL each all targets for all except targets for except LPA (18:1)for LPA at 17 (18:1) ng/ µatL 17 and ng/ PAμL (36:2) and PA at 2 (36:2) ng/µ L.at (2B ng/) RepresentativeμL. (B) Representative precursor precursorion mass spectra ion mass obtained spectra by obtained tandem mass by tandem tag (TMT) mass reporter tag (TMT) ion-speciﬁc reporter precursor ion-specific ion scanning precursor analysis. ion scanning Injection analysis. volume Injectionwas 5 µL volume and the was concentration 5 μL and ofthe standards concentration for derivatization of standards wasfor derivatization 42.5 pg /µL each was for 42.5 all pg targets /μL excepteach for for all LPA(18:1) targets ex- at cept17 ng/ forµ LLPA(18:1) and PA(36:2) at 17 at ng/ 2 ng/μL andµL. EicosanoidsPA(36:2) at 2 and ng/ AAμL. wasEicosanoids derivatized and with AA aminoxywas deriva TMT-128tized with and aminoxy precursor TMT-128 ion scanning and precursor ion scanning for 128.15 was performed. LPA (18:1) and PA (36:2) was derivatized with aminoxy TMT-129 and for 128.15 was performed. LPA (18:1) and PA (36:2) was derivatized with aminoxy TMT-129 and precursor ion scanning for precursor ion scanning for 129.15 was performed. Positive ion mode and collision energy at −45 eV were used. Only the 129.15 was performed. Positive ion mode and collision energy at −45 eV were used. Only the mass spectra at the retention mass spectra at the retention time indicated for the target was shown. time indicated for the target was shown. 11,15-Dioxo-9α-hydroxy-2,3,4,5-tetranorprostan-1,20-dioic acid (Tetranor PGDM) 11,15-Dioxo-9α-hydroxy-2,3,4,5-tetranorprostan-1,20-dioic acid (Tetranor PGDM) and 20-carboxy Leukotriene B4 (LTB4) haveand 20-carboxy two carboxy Leukotriene groups to be B4 derivatized,(LTB4) have and two divalent carboxy ions groups were to bedetected. derivatized, Note that and RT divalent 13.11-13.18 ions were and detected.RT 13.38-13.29 Note showedthat RT 13.11-13.18unexpected and m/ RTz (in 13.38-13.29 parenthesis). showed Mass unexpected chromatogramsm/z (in parenthesis).for those ions Mass were chromatograms not identicalfor to thoseTMT-TXB2 ions were or TMT-20-carboxynot identical to TMT-TXB2 LTB4. (C) orRepresentative TMT-20-carboxy product LTB4. ion (C) mass Representative spectra obtain producted by ion product mass spectra ion scans obtained of derivatized by product com- ion pounds.scans of derivatizedConcentration compounds. of compounds, Concentration used TMT of compounds,reporters and used injection TMT reporters volume were and injection same as volume (B). Precursor were same ion as(m (/Bz).) andPrecursor collision ion energy (m/z) and(eV) collision were indicated. energy (eV) were indicated.

2.3. Reproducibility 2.3. Reproducibility Next, the reproducibility of TMT labeling was confirmed. Authentic standards of PA, LPA,Next, and the eicosanoid-related reproducibility of fatty TMT acids labeling were was added confirmed. to phosphate Authentic buffer standardssaline (PBS) of orPA, human LPA, andpooled eicosanoid-related serum at 2-, 4-, 8-, fatty and acids 16-fol wered dilutions, added to and phosphate 1-buthanol buffer extraction saline (PBS) was performedor human pooledin 0.7 M serum sodium at 2-, chloride. 4-, 8-, andThe 16-foldrepeatability dilutions, of TMT and 1-buthanolderivatization extraction in the presencewas performed or absence in 0.7 of M serum sodium was chloride. satisfacto Thery, repeatability with a coefficient of TMT of derivatization variation ranging in the frompresence 1.8% or to absence 28.4%, as of shown serum wasin Table satisfactory, 1. The reproducibility with a coefficient was of slightly variation better ranging in serum from than1.8% in to phosphate 28.4%, as shown buffer. in The Table reason1. The for reproducibility this might be that was lipids slightly are better difficult in serumto mix thanwith PBSin phosphate and that adsorption buffer. The to reason the container for this might is a concern; be that lipidstherefore, are difficultexperiments to mix with with lipids PBS and that adsorption to the container is a concern; therefore, experiments with lipids in the in the presence of serum could have better reproducibility. The relative linearity between presence of serum could have better reproducibility. The relative linearity between the the area value of TMT-derived targets and the sample dilution factor of the TMT-labeled area value of TMT-derived targets and the sample dilution factor of the TMT-labeled lipids lipids ranged from 0.940 to 0.999 as a correlation coefficient, regardless of the presence or ranged from 0.940 to 0.999 as a correlation coefficient, regardless of the presence or absence absence of serum. From the results of assay validation shown in Table 1, there were no

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of serum. From the results of assay validation shown in Table1, there were no degradation reactions or other factors that would affect the reproducibility. Our reaction conditions are very mild in terms of pH and temperature, however, there may be slightly unstable lipid molecules during derivatization, and they do not affect the relative quantiﬁcation because they are derivatized together and normalized by the bridging sample measured simultaneously.

Table 1. Reproducibility of peak area obtained by tandem mass tag (TMT) derivatization methodology in different concentrations of standard lipids.

12-HETE × 16 12-HETE × 8 12-HETE × 4 12-HETE × 2 12-HETE × 1 Correlation CV% in Buffer 8.04 13.03 13.36 4.42 5.26 0.966 CV% in Serum 4.18 8.23 4.60 2.93 1.78 0.951 20-HETE × 16 20-HETE × 8 20-HETE × 4 20-HETE × 2 20-HETE × 1 Correlation CV% in Buffer 6.86 8.51 11.01 5.79 4.03 0.963 CV% in Serum 4.01 8.54 3.01 2.71 2.99 0.949

PGE2 × 16 PGE2 × 8 PGE2 × 4 PGE2 × 2 PGE2 × 1 Correlation CV% in Buffer 5.14 8.97 8.20 4.34 5.91 0.962 CV% in Serum 4.01 8.54 3.01 2.71 2.99 0.949

TXB2 × 16 TXB2 × 8 TXB2 × 4 TXB2 × 2 TXB2 × 1 Correlation CV% in Buffer 7.93 10.18 12.41 4.91 6.14 0.999 CV% in Serum 4.81 6.65 5.86 7.51 7.75 0.998

20-carboxy-LTB4 20-carboxy-LTB4 20-carboxy-LTB4 20-carboxy-LTB4 20-carboxy-LTB4 × 16 × 8 × 4 × 2 × 1 Correlation CV% in Buffer 11.30 10.59 27.28 14.91 12.27 0.994 CV% in Serum 15.26 6.23 5.42 5.70 11.09 0.996 tetranor-PGDM tetranor-PGDM tetranor-PGDM tetranor-PGDM tetranor-PGDM × 16 × 8 × 4 × 2 × 1 Correlation CV% in Buffer 18.83 14.52 28.35 14.87 12.62 0.996 CV% in Serum 6.69 9.37 7.06 9.13 10.40 0.978 LPA (16:0) × 16 LPA (16:0) × 8 LPA (16:0) × 4 LPA (16:0) × 2 LPA (16:0) × 1 Correlation CV% in Buffer 22.15 17.68 16.81 16.69 8.02 0.998 CV% in Serum 8.40 9.57 9.13 10.64 2.79 0.998 LPA (18:1) × 16 LPA (18:1) × 8 LPA (18:1) × 4 LPA (18:1) × 2 LPA (18:1) × 1 Correlation CV% in Buffer 16.32 15.21 19.61 9.43 7.52 0.998 CV% in Serum 6.18 9.55 12.70 7.47 4.79 0.989 PA (36:2) × 16 PA (36:2) × 8 PA (36:2) × 4 PA (36:2) × 2 PA (36:2) × 1 Correlation CV% in Buffer - 12.29 12.55 5.27 4.03 0.999 CV% in Serum 2.55 7.21 5.23 5.22 2.02 0.995 Standard lipids were added to PBS buffer (n = 3) or human serum (n = 4). The final concentration in ×1 sample was 170 ng/mL for 12-hydroxyeicosatetraenoic acid (12-HETE), 20-hydroxyeicosatetraenoic acid (20-HETE), thromboxane B2 (TXB2), prostaglandin E2 (PGE2), arachidonic acid (AA), tetranor-PGDM, and 20-carboxy-LTB4, 0.9 µM for lysophosphatidic acid (LPA)(16:0) and LPA(18:1), and 0.47 µg/mL for phosphatidic acid (PA)(18:1/18:1). Serial 2-fold dilutions of standard mixtures were performed to prepare ×2, ×4, ×8, and ×16 diluted standards. The coefficient of variation (CV) for area value in each peak area of PBS buffer (n = 3) or human serum (n = 4) samples was calculated. The correlation coefficient between the dilution factor and peak area was calculated using the CORREL function add-in in Excel 2016.

2.4. Stability of Phosphatidic Acid (PA) in Serum During reproducibility experiments using serum samples, we were unable to detect any endogenous PAs in the serum. Although the sensitivity of this study depends partly on the MS performance and HPLC conditions, the stability of PA in the serum was investigated by adding a PA standard to the human pooled serum. We observed a rapid decrease in PA added to the serum (Figure4 (left)) and an increase in LPA instead (Figure4 (right)). This is consistent with the fact that serum LPA levels are already known to increase during serum incubation [25,29]. To examine the stability of several other lipid signaling molecules MetabolitesMetabolites 2021 2021, 11, ,x 11 FOR, x FOR PEER PEER REVIEW REVIEW 7 of 715 of 15

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4 (right)).4 (right)). This This is consistent is consistent with with the thefact fact that that serum serum LPA LPA levels levels are arealready already known known to in- to in- creasecrease during during serum serum incubation incubation [25,29]. [25,29]. To Toexamine examine the thestability stability of severalof several other other lipid lipid thatsignalingsignaling were notmolecules molecules labeled that with that were TMT, were not they not labeled were labeled measuredwith wi TMT,th TMT, using they they thewere were same measured measured pooled using serum using the sample thesame same (serumpooledpooled supplementedserum serum sample sample with(serum (serum PA). supplemented LPCsupplemented is thought with with to PA). be PA). a LPC source LPC is thought ofis LPAthought viato beto action abe source a ofsource the of of extracellularLPALPA via viaaction enzymeaction of theof autotaxin, theextracellular extracellular and theenzyme increaseenzyme autotaxin, inautotaxin, LPC during and and the serum theincrease increase incubation in LPCin LPC has during alsoduring beenserumserum reported incubation incubation [28]. has As has also expected, also been been reported LPC reported (18:1) [28] [28]. tendedAs. Asexpected, expected, to increase LPC LPC (18:1) in (18:1) the tended serum tended to (Figure increase to increase5 (left)).in thein the Inserum contrast, serum (Figure (Figure lysophosphatidylethanolamine 5 (left)). 5 (left)). In contrast,In contrast, lysophosphatidylethanolamine lysophosphatidylethanolamine (LPE) (18:1) did not change (LPE) significantly(LPE) (18:1) (18:1) did did (notFigurenot change5 change(middle)) significantly significantly and DAG (Figure (18:1/18:1)(Figure 5 (middle)) 5 (middle)) appeared and toand DAG decrease DAG (18:1/18:1) slightly(18:1/18:1) inappeared the appeared serum to ( Figuredecreaseto decrease5 (right)).slightlyslightly in These thein theserum results serum (Figure suggest (Figure 5 (right)). that 5 (right)). the These stability These results ofresults the suggest bioactivesuggest that that lipidsthe thestability in stability serum of theof varies thebio- bio- dependingactiveactive lipids lipids on in the serum in molecular serum varies varies species.depending depending on theon themolecular molecular species. species.

Figure 4. Stability of phosphatidic acid (PA) (36:2) in human serum during 37 ◦C incubation. A standard stock of PA FigureFigure 4. Stability 4. Stability of phosphatidicof phosphatidic acid acid (PA) (PA) (36:2) (36:2) in human in human serum serum during during 37 °C37 incubation.°C incubation. A standard A standard stock stock of PAof PA (18:1/18:1) was dissolved in serum and incubated for the indicated time. Two replicate samples with 5.1 ng of PA in (18:1/18:1)(18:1/18:1) was was dissolved dissolved in serum in serum and and incubated incubated for thefor theindi indicatedcated time. time. Two Two replicate replicate samples samples with with 5.1 ng5.1 ofng PA of PAin 30 in μ 30L μL 30 µL pooled serum were tested. (left) PA (36:2) and (right) LPA (18:1) were analyzed using liquid chromatography/mass pooledpooled serum serum were were tested. tested. (left ()left PA) PA(36:2) (36:2) and and (right (right) LPA) LPA (18:1) (18:1) were were analyzed analyzed using using liquid liquid chromatography/mass chromatography/mass spectrometryspectrometryspectrometry (LC/MS)(LC/MS) (LC/MS) after after lipidlipid lipid extraction.extraction. extraction. The The X-axis-axis X-axis showsshows shows incubationincubation incubation timetime time (h)(h) andand(h) and thethe Y theY-axis-axis Y-axis showsshows shows valuesvalues values relative relative relative valuesvaluesvalues to to those those to those at at 0 0 h. ath. Two0Two h. Two replicate replicate replicate sample samp sample results resultsle results (#1 (#1 and and(#1 #2)and #2) are are#2) shown areshown shown as as lines. lines. as lines.

Figure 5. Stability of endogenous lysophospholipids and diacylglycerol in human serum during 37 ◦C incubation. PooledFigureFigure serum5. Stability 5. Stability was of incubated endogenous of endogenous for thelysophospholipids indicatedlysophospholipids time and and diacylglyc (left diacylglyc) lysophosphatidylcholineerolerol in human in human serum serum during (LPC) during 18:1,37 °C 37 ( middleincubation. °C incubation.) lysophos- Pooled Pooled phatidylethanolamineserumserum was was incubated incubated (LPE)for thefor 18:1, theindicated indicated and ( righttime time )and diacylglycerol and (left ()left lysophosphatidylcholine) lysophosphatidylcholine (DAG) 18:1/18:1 were (LPC) (LPC) analyzed 18:1, 18:1, (middle using (middle) LC/MS lysophosphatidyl-) lysophosphatidyl- after lipid extraction.ethanolamineethanolamine The (LPE)X-axis (LPE) 18:1, shows 18:1, and incubationand (right (right) diacylglycerol) timediacylglycerol and values (DAG) (DAG) are 18:1/18:1 shown 18:1/18:1 onwere the were analy-axis analyzed relativeyzed using using to LC/MS those LC/MS atafter 0 after h. lipid Two lipid extraction. replicate extraction. The X-axis shows incubation time and values are shown on the y-axis relative to those at 0 h. Two replicate sample results sampleThe results X-axis areshows shown incubation as lines. time and values are shown on the y-axis relative to those at 0 h. Two replicate sample results are areshown shown as lines. as lines.

2.5.2.5.2.5. StabilityStability Stability ofof FattyFatty of Fatty AcidsAcids Acids andand and TheirTheir Their MetabolitesMetabolites Metabolites inin SerumSerum in Serum WeWe collectedWecollected collected 172172 serum172serum serum samplessamples samples fromfrom from patients patients patients over over over 62 62years years62 yearsof ofage age of age withwith with ﬁvefive five diseasesdiseases diseases (Alzheimer’s(Alzheimer’s(Alzheimer’s disease,disease, disease, Parkinson’s Parkinson’s Parkinson’s disease, disease, disease, depression, depression, depression, schizophrenia, schizophreni schizophreni anda, anda, stroke) and stroke) stroke) from from the from Universitythe theUniversity University of Tokyo of Tokyo of Hospital Tokyo Hospital Hospital (Table (Table2). (Table 2). 2).

TableTable 2. Demographic 2. Demographic characteristics characteristics of patients. of patients.

Parkinson’sParkinson’s Dis- Dis- Alzheimer’sAlzheimer’s Disease Disease DepressionDepression SchizophreniaSchizophrenia StrokeStroke easeease N N 67 67 26 26 21 21 21 21 37 37

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Table 2. Demographic characteristics of patients. Female % 62.7% 53.8% 52.4% 42.9% 37.8% Alzheimer’s Disease Depression Parkinson’s Disease Schizophrenia Stroke Age (years) N 80.3 ± 7.18 67 75.3 ± 6.70 26 74.4 ± 5.50 21 72.7 ± 5.82 21 77.5 ± 376.07 mean ± SD Age (years)Female % 62.7% 53.8% 52.4% 42.9% 37.8% 62–95 65–88 66–90 65–82 65–90 AgeRange (years) mean ± SD 80.3 ± 7.18 75.3 ± 6.70 74.4 ± 5.50 72.7 ± 5.82 77.5 ± 6.07 Age (years) Range 62–95 65–88 66–90 65–82 65–90 The usefulness of serum lipid analysis has already been reported with respect to severalThe aspects usefulness [30,31]. of serum To screen lipid analysisthe trends has in already the difference been reported in the with stability respect of toserum several li- pidsaspects in [a30 variety,31]. To of screen diseases, the trends we tested in the differencea pooled inserum the stability mixture of of serum the same lipids amount in a variety of individualof diseases, serum we tested from a pooledpatients serum with mixtureeach disease. of the We same also amount random of individually selected serum 300 serum from samplespatients withas pooled each disease.QC samples We also among randomly approximately selected 3006000 serum patients samples (average as pooled age: ~60, QC female:samples ~50%) among who approximately visited the 6000University patients of (average Tokyo Hospital age: ~60, for female: disease ~50%) treatment who visited and diagnosis.the University We performed of Tokyo Hospital TMT la forbeling disease of six treatment different and report diagnosis.er ions Webased performed on six differ- TMT entlabeling pooled of sixserum different samples reporter from ionsa total based of five on sixdiseases different and pooled a QC sample, serum samples that is, froma sim- a ultaneoustotal of five comparison diseases and of asix QC different sample, TMT-labeled that is, a simultaneous samples in comparison a single LC/MS of six differentrun. To testTMT-labeled the stability samples of serum in a single lipids, LC/MS we used run. an To incubation test the stability temperature of serum of lipids, 37 °C, we which used anis similarincubation to that temperature inside the of body. 37 ◦C, The which level is similarrelative to to that that inside of the the QC body. pooled The levelserum relative and the to stabilitythat of the of QC endogenous pooled serum eicosanoids, and the stability fatty acids, of endogenous and LPA eicosanoids,in each disease fatty serum acids, and sample LPA arein each shown disease in Figure serum 5. sample Regarding are shown free infatty Figure acid5. (FA), Regarding FA (18:1), free fatty and acid FA (FA),(20:1) FA (Figure (18:1), 6A,B),and FA the (20:1) level (Figure between6A,B), before the level and betweenafter incubation before and did after not incubationseem to vary did significantly not seem to amongvary significantly diseases, whereas among diseases, arachidonic whereas acid arachidonic (AA, FA (20:4)) acid (AA, increased FA (20:4)) in all increased six samples in all (Figuresix samples 6C), (Figureand a similar6C), and trend a similar was observed trend was for observed eicosapentaenoic for eicosapentaenoic acid (EPA, acidFA (20:5)) (EPA, (FigureFA (20:5)) 6D) (Figure and docosahexaenoic6D) and docosahexaenoic acid (DHA, acid FA (DHA, (22:6)) FA (Figure (22:6))(Figure 6E), whereas6E), whereas FA (22:4) FA did(22:4) not did change not change significantly significantly (Figure (Figure 6F). 6F).

Figure 6. Cont.

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Figure 6. Stability of endogenous free fatty acids in serum. Pooled quality control (QC) serum ◦ Figuresamples 6. andStability pooled of endogenous disease serum free samples fatty acids were in incubatedserum. Pooled at 37 qualityC for 20control h. Three (QC) replicates serum of sampleseach pooled and pooled serum sampledisease wereserum tested. samples Extraction, were incubated tandem at mass37 °C tagfor 20 (TMT) h. Three derivatization, replicates of and eachLC/MS pooled analysis serum were sample performed were tested. as described Extraction, in the tandem Methods mass section. tag (TMT) The Y derivatization,-axis shows the and values LC/MSrelative analysis to those were of pooled performed QC serum as described with no in incubation the Methods (0 h) section. for each The lipid. Y-axis Error shows bars the represent values relativethe standard to those error of pooled of the mean. QC serum FA, fattywith acid;no incu AA,bation arachidonic (0 h) for acid;each lip EPA,id. eicosapentaenoicError bars represent acid; the standard error of the mean. FA, fatty acid; AA, arachidonic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid. (A) FA (18:1), (B) FA (20:1), (C) AA/FA (20:4), (D) EPA/FA (20:5), DHA, docosahexaenoic acid. (A) FA (18:1), (B) FA (20:1), (C) AA/FA (20:4), (D) EPA/FA (20:5), (E) (E) DHA/FA (22:6), (F) FA (22:4). DHA/FA (22:6), (F) FA (22:4). Despite the approximate chemical structure, the cause of the differences in stability amongDespite the different the approximate molecular specieschemical is notstructure, likely tothe be cause a simple of the oxidation differences or light-induced in stability amongreaction. the Group different 10 secretory molecular phospholipase species is A2not (PLA2G10) likely to isbe known a simple to hydrolyze oxidation phos- or light-inducedpholipids and reaction. shows an Group apparent 10 preferencesecretory forphospholipase PUFAs, such A2 as EPA,(PLA2G10) DPA,and is known AA, with to hydrolyze phospholipids and shows an apparent preference for PUFAs, such as EPA,

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DPA,respect and to substrateAA, with phospholipids respect to substrate [32]. The phospholipids results indicate [32]. that The PLA2G10 results mightindicate be that acti- PLA2G10vated in freeze-thawed might be activated serum. in For freeze-thawed eicosanoids, 12-HETEserum. For (hydroxyeicosatetraenoic eicosanoids, 12-HETE acid)(hy- droxyeicosatetraenoictended to increase (Figure acid)7 A)tended after to incubation, increase (Figure for example, 7A) after whereas incubation, 20-/18-/15-HETE for example, whereastended to 20-/18-/15-HETE decrease in serum tended levels to (Figuredecrease7B). in Sinceserum 12-HETE levels (Figure and 15-HETE 7B). Since are 12-HETE down- andstream 15-HETE of lipoxygenase are downstream enzymes of and lipoxygenase 18-HETE and enzymes 20-HETE and are 18-HETE produced and by a20-HETE cytochrome are producedP450 enzyme, by a itcytochrome is not surprising P450 thatenzyme, the activityit is not of surprising these enzymes that the is inﬂuenced activity of by these diseases [33]. In disease samples, thromboxane B2 (TXB2) tended to decrease in serum after enzymes is influenced by diseases [33]. In disease samples, thromboxane B2 (TXB2) incubation, especially for patients with schizophrenia (Figure7C). One type of molecule tended to decrease in serum after incubation, especially for patients with schizophrenia that appears to play an important role in this disease is eicosanoids such as TXB [34]. As (Figure 7C). One type of molecule that appears to play an important role in this disease2 is already reported in papers on LPA [23,29,35], LPA (18:2) was observed in serum and it was eicosanoids such as TXB2 [34]. As already reported in papers on LPA [23,29,35], LPA increased by incubation (Figure7D). (18:2) was observed in serum and it was increased by incubation (Figure 7D).

Figure 7. Cont.

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DPA, and AA, with respect to substrate phospholipids [32]. The results indicate that PLA2G10 might be activated in freeze-thawed serum. For eicosanoids, 12-HETE (hydroxyeicosatetraenoic acid) tended to increase (Figure 7A) after incubation, for example, whereas 20-/18-/15-HETE tended to decrease in serum levels (Figure 7B). Since 12-HETE and 15-HETE are downstream of lipoxygenase enzymes and 18-HETE and 20-HETE are produced by a cytochrome P450 enzyme, it is not surprising that the activity of these enzymes is influenced by diseases [33]. In disease samples, thromboxane B2 (TXB2) tended to decrease in serum after incubation, especially for patients with schizophrenia (Figure 7C). One type of molecule that appears to play an important role in this disease is eicosanoids such as TXB2 [34]. As already reported in papers on LPA [23,29,35], LPA (18:2) was observed in serum and it was increased by incubation (Figure 7D).

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FigureFigure 7.7. StabilityStability ofof endogenousendogenous eicosanoidseicosanoids and and LPA LPA in in serum. serum. Pooled Pooled quality quality control control (QC) (QC) serum se- samplesrum samples and pooledand pooled disease disease serum serum samples samples were were incubated incubated at 37 at◦ 37C for°C for 20 h.20 Threeh. Three replicates replicates of eachof each pooled pooled serum serum sample sample were were tested. tested. Extraction, Extraction, tandemtandem massmass tagtag (TMT) derivatization, derivatization, and and LC/MSLC/MS analysis were performed as de describedscribed in the Methods section. The Y-axis-axis shows the values relativerelative toto thosethose of of pooled pooled QC QC serum serum with with no no incubation incubation (0 (0 h) h) for for each each lipid. lipid. Error Error bars bars represent represent the the standard error of the mean (SEM). HETE, hydroxyeicosatetraenoic acid; TXB2, thromboxane standard error of the mean (SEM). HETE, hydroxyeicosatetraenoic acid; TXB2, thromboxane B2, LPA, B2, LPA, lysophosphatidic acid. (A) 12-HETE, (B) mixture of 20-HETE/18-HETE/15-HETE, or one lysophosphatidic acid. (A) 12-HETE, (B) mixture of 20-HETE/18-HETE/15-HETE, or one of them, of them, (C) TXB2, (D) LPA (18:2). (C) TXB2,(D) LPA (18:2). We detected a number of unknown peaks with TMT labels that could not be identi- We detected a number of unknown peaks with TMT labels that could not be identiﬁed asfied molecular as molecular species. species. Representative Representative examples examples of their of their stability stability in serum in serum are are shown shown in Figurein Figure8. The8. The unknown unknownm /mz/z622.55 622.55 peak peak showed showed a a decrease decreasein in serumserum levelslevels forfor thethe pooledpooled Parkinson’sParkinson’s diseasedisease samplesample (Figure(Figure8 8A),A), but but others others did did not not show show such such reductions. reductions. ApartApart fromfrom serumserum stability,stability, anan unknown unknownm m//zz 656.55656.55 peakpeak (Figure(Figure8 8B)B) was was detected detected as as a a higherhigher signalsignal in in stroke stroke patient patient serum serum among among other other diseases. diseases. The unknownThe unknownm/z 622.55 m/z 622.55 peak waspeak set was up set as theup monitoringas the monitoring channel channel for HETEs for andHETEs epoxyeicosatrienoic and epoxyeicosatrienoic acids (EETs), acids eicosanoid(EETs), eicosanoid subcategories subcategories with number with of numb structuraler of isomers structural (i.e., isomers 5-HETE, (i.e., 8-HETE, 5-HETE, and 11-HETE,8-HETE, and among 11-HETE, others, among for HETEs others, or 5(6)-EET,for HETE 8(9)-EET,s or 5(6)-EET, and 14(15)-EET,8(9)-EET, and among 14(15)-EET, others, among others, for EET). The unknown m/z 656.55 peak corresponded to prostaglandins for EET). The unknown m/z 656.55 peak corresponded to prostaglandins D1 and E1, 1 1 2 prostaglandinD and E , prostaglandin F2, and their F isomers., and their Because isomers. the Because standard the compounds standard derivatizedcompounds withderi- TMTvatized were with not TMT fully were separated not fully from separated each other, from we dideach not other, conﬁrm we did these not peaks confirm as one these of thesepeaks candidates. as one of these candidates.

Figure 8. Stability of endogenous unknowns in serum.Pooled quality control (QC) serum samples and pooled disease serumFigure samples 8. Stability were of incubatedendogenous at 37unknowns◦C for 20 in h. serum. Three replicatesPooled quality of each control pooled (QC) serum serum sample samples were and tested. pooled Extraction, disease tandemserum samples mass tag were (TMT) incubated derivatization, at 37 °C and for LC/MS20 h. Three analysis replicates were of performed each pooled as described serum sample in the were Methods tested. section. Extraction, The Ytandem-axis shows mass the tag values (TMT) relative derivatization, to those ofand pooled LC/MS QC analysis serum with were no performed incubation as (0 described h) for each in lipid. the Methods Error bars section. represent The theY-axis standard shows error the values of the meanrelative (SEM). to those (A) of Unknown pooled QC peak serum at m /withz 622.55, no incubation (B) Unknown (0 h) peakfor each at m lipid./z 656.55. Error bars represent the standard error of the mean (SEM). (A) Unknown peak at m/z 622.55, (B) Unknown peak at m/z 656.55. It might be possible to isolate all eicosanoids that are the same or close in molecular weightIt andmight identify be possible each peak to isolate withvery all eicosanoids sophisticated that HPLC are the conditions. same or However,close in molecular in many cases,weight HPLC and separationidentify each is a peak time-consuming with very sophisticated procedure that HPLC conﬂicts conditions. with high-throughput However, in multi-specimenmany cases, HPLC analysis, separation which isis characteristic a time-consuming of the TMT procedure approach. that Therefore, conflicts ifwith an interestinghigh-throughput candidate multi-specimen peak is found analysis, by high-throughput which is characteristic multi-specimen of the TMT analysis, approach. it is followedTherefore, by if another an interesting analytical candidate method peak focusing is found on that by peakhigh-throughput for compound multi-specimen identiﬁcation. analysis, it is followed by another analytical method focusing on that peak for compound identification. Apart from peak identification, the stability and expression levels of these substances in the serum might differ depending on the type of lipid or disease. In this study, we developed a multi-specimen analysis for serum lipids and a relative quantification method using a pooled QC sample with TMT labeling. Stability of the lipids in human serum samples were examined. Stability was different among lipid species and also among samples, and we were reminded of the importance of process control from blood collection to serum preparation and storage.

3. Materials and Methods

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Apart from peak identiﬁcation, the stability and expression levels of these substances in the serum might differ depending on the type of lipid or disease. In this study, we developed a multi-specimen analysis for serum lipids and a relative quantiﬁcation method using a pooled QC sample with TMT labeling. Stability of the lipids in human serum samples were examined. Stability was different among lipid species and also among samples, and we were reminded of the importance of process control from blood collection to serum preparation and storage.

3. Materials and Methods 3.1. Reagents AminoxyTMT sixplex Label Reagent Set was purchased from Thermo Fisher Sci- entiﬁc Inc. (Waltham, MA, USA). Phospholipid mixtures were obtained from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). Lipid mediator standards, 12-HETE, 20-HETE, TXB2, PGE2, arachidonic acid, tetranor-PGDM, and 20-carboxy-LTB4 were obtained from Cayman Chemical (Ann Arbor, MI, USA). LPA (16:0), LPA (18:1), and PA (36:2) were obtained from Avanti Polar Lipids (Alabaster, AL, USA). DMTMM and 4-methylmorpholine (NMM) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). 1-Ethyl-3-[3- dimethylaminopropyl]carbodiimide hydrochloride (EDC) was obtained from Fuji Film Wako Pure Chemical Co. (Osaka, Japan). Special grade reagents of ammonium bicarbonate, sodium chloride and 1-buthanol, and HPLC grade solvents of methanol, isopropanol and acetonitrile were purchased from Wako Pure Chemical Co. The authentic lipid standard stock solutions were 12-HETE (0.6 µg/mL), 20-HETE (0.6 µg/mL), TXB2 (0.6 µg/mL), PGE2 (0.6 µg/mL), AA (0.6 µg/mL), tetranor-PGDM (0.6 µg/mL), 20-carboxy-LTB4 (0.6 µg/mL), LPA(16:0) (10 µM), LPA (18:1) (10 µM), PA (36:2) (5 µg/mL).

3.2. Samples This study was conducted with the approval of the ethics committee of the University of Tokyo Hospital in compliance with the relevant guidelines and regulations. A QC sample was generated by pooling 300 patient residual samples after the completion of the requested clinical laboratory tests at the University of Tokyo Hospital (the approval number by the Institutional Research Ethics Committee of the Faculty of Medicine, University of Tokyo, was 2019022NI). The Japanese neurodegenerative and psychiatric disease specimens were obtained from residue samples from patients who had been examined at the University of Tokyo Hospital during a 3-week period (the approval number by the Institutional Research Ethics Committee of the Faculty of Medicine, The University of Tokyo, was 2019068NI). Informed consent for participation in the study was obtained using an opt-out process on the web page of The University of Tokyo Hospital. The investigations were carried out following the rules of the Declaration of Helsinki of 1975 (https://www.wma.net/ what-we-do/medical-ethics/declaration-of-helsinki/), revised in 2013. Diagnosis in the electronic medical record system recorded within 2 years before the date of blood sampling was used. All samples were stored at −80 ◦C prior to analysis.

3.3. Lipid Extraction We added 15 µL of 2 M NaCl to 30 µL of serum and mixed it well. Next, 240 µL of 1-butanol was added to the sample, mixed, and then centrifuged at 10,000× g for 2 min at 4 ◦C, and 200 µL of the supernatant was transferred into a new tube, and the solvent was allowed to evaporate using a vacuum evaporator (EC-95C3T, SAKUMA, Tokyo, Japan). In the experiment on extraction, the results of LC/MS analysis without extraction were compared with the results after extraction, but in this case, the comparison with the addition of standards, but without TMT derivatization.

3.4. TMT Reaction The samples after extraction and drying were resuspended in 20 µL methanol in the tube. Then, 20 µL of 1% NMM in chloroform was added and mixed. Next, 10 µL of Metabolites 2021, 11, 19 13 of 15

33.3 mg/mL EDC in methanol was added to the sample tube and 40 µL of aminoxyTMT dissolved in 200 µL of acetonitrile was mixed. Then, 10 µL of 10 mg/mL DMTMM in methanol was added and the mixture was allowed to react at room temperature for 24 h. Finally, 5 µL of 10% acetone in methanol was added to quench the unreacted reagent. After completion of TMT derivatization, equal amounts were mixed to form one set of six TMT reporter ions (numbers 126–131) to make one vial.

3.5. Liquid Chromatography/Mass Spectrometry (LC/MS) Measurements The LC and MS measurement conditions were as follows. The sixplex aminoxyTMT™ derivatized samples were subjected to the Nexera UHPLC system and LCMS-8040 or -8060 triple quadrupole mass spectrometers (Shimadzu Co., Kyoto, Japan). The aminoxyTMT derivatized samples were diluted twice with methanol before measurement. An Acquity UPLC BEH C8 column (1.7 µm, 2.1 × 100 mm; Waters) was used with the following mobile phase compositions: 5 mM NH4HCO3/water (mobile phase A), acetonitrile (mobile phase B), and isopropanol (mobile phase C). The pump gradient was programmed as follows [time (%A/%B/%C)]: 0 min (95/5/0), to 8 min (70/30/0), 16 min (30/35/35), 28 min (6/47/47), 35 min (6/47/47), 35.1 min (95/5/0); it was then held for 38 min for equilibra- tion. The ﬂow rate was 0.35 mL/min, and column temperature was 47 ◦C. The injection volume was 5 µL. SRM analysis was performed using the positive ion mode ESI, with a collision energy of 46 eV for TMT-derivatized compounds. For the sixplex aminoxyTMT sample, Fatty acid species possessing carbon chains 12 to 24 carbons in length were tar- geted [22], 132 eicosanoids or other fatty acid derivatives [36], ﬁve LPA species (16:0, 18:0, 18:1, 18:2, 20:4), and nine PA species (32:0, 34:1, 34:2, 36:0, 36:2, 36:3, 36:2, 36:4, 38:4) were tar- geted. The selective reaction monitoring (SRM) transitions for TMT-derivatized molecules, [M-H+303.25]+ →126.15, →127.15, →128.15, →129.15, →130.15, and →131.15 were used. The SRM transitions for LPC (16:0), LPE (18:1), and DAG (18:1/18:1), 496.35 → 184.1, 480.3 → 339.3, and 636.6 → 339.3, respectively, in positive ion mode. The SRM transitions for non-labeled LPA (16:0), LPA (18:1), and PA (36:2), were 409.4 → 153.1, 435.4 → 153.1, and 699.6 → 153.1, respectively, in negative ion mode. The SRM settings for non-labeled eicosanoids have been described previously [36]. All data were analyzed using Microsoft Excel 2016.

4. Conclusions In the TMT method, six samples are mixed into one and analyzed by LC/MS as a single set. Since six LC/MS measurements can be completed in a single run, a six-fold increase in sample throughput can be achieved compared to conventional methods. This is one of the merits, and another advantage is quantitation. The TMT method does not require internal standards, which have to be obtained and prepared depending on the number of targets. Instead, a common bridging sample is prepared to normalize data. One of the six samples is a bridging sample (pooled QC sample) that is common among the sets. Since we perform relative quantification to this bridging sample, we can obtain reliable quantification values as long as we use the bridging sample. We have made it possible to derivatize lipid molecules with carboxy groups and lipid molecules with phosphate groups at the lipid ends simultaneously by TMT. However, there are many lipid molecules that cannot be TMT-derivatized. Therefore, it is necessary to develop a new TMT method to expand the range of application. We mixed 300 individual serum samples as a pooled QC sample to make a common bridging sample. The disadvantage of this method is that relative quantification is not possible for peaks that are not detected in the pooled QC samples. In serum preparations, blood samples are kept at room temperature for 30–60 min after collection to allow clots to form and remove cells, clotting factors, and other cellular components. It is easy to imagine from our results that the inadequate control of temperature and time in this step would lead to large variations in data [37–39]. Therefore, proper sample collection, preparation, and storage are important to obtain reliable outcomes. Metabolites 2021, 11, 19 14 of 15

However, the association between the disease and serum lipid stability is of great interest because various factors, such as enzyme activity affected by the disease situation in serum, might affect the stability. We believe that the method developed here can be applied to the analysis of many clinical samples, not only for serum samples but also organ samples and various cell samples. The TMT labeling method is expected to be one of the most useful analytical methods for bioactive lipids and lipidomics.

Author Contributions: Conceptualization, Y.O.; methodology and data analysis, S.M.T.; software, Y.K.; clinical samples, M.S. and Y.Y.; writing—original draft preparation, S.M.T. and Y.O.; writing— review and editing, T.S. and Y.Y.; supervision, T.S. and Y.Y.; funding acquisition, S.M.T., T.S. and Y.O. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by AMED, Japan Agency for Medical Research and Development, grant numbers JP19ae0101078s0301 and 20ae0101078s0302 (Grants-in Aid from Project Focused on Developing Key Technology for Discovering and Manufacturing Drugs for Next-Generation Treatment and Diagnosis, Study research for the construction of ecosystems concerning practical applications of drug discovery technology seeds.). The Lipidomics Laboratory of the University of Tokyo is supported by the Shimadzu Corporation. Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the ethics committee of the University of Tokyo Hospital in compliance with the relevant guidelines and regulations (protocol codes were 2019022NI and 2019068NI). Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: The data presented in this study are available in article. Acknowledgments: We thank Chiyo Morita at the University of Tokyo for technical assistance in lipid analysis. Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript; or in the decision to publish the results.

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