Tri-Reagent Homogenate Is a Suitable Starting Material for UHPLC-MS Lipidomic Analysis

Ci a ion: F esnedo, O.; Abad-Ga cia, B.; Rueda, Y. T i-Reagen Homogena e Is a Sui able S a ing Ma e ial o UHPLC-MS Lipidomic Analysis. Sepa a ions 2022,9, 268. h ps://doi.o g/10.3390/ sepa a ions9100268 Academic Edi o s: P ee i Chand a and A ul Singh Ra ho e Recei ed: 1 Augus 2022 Accep ed: 15 Sep embe 2022 Published: 27 Sep embe 2022 Publishe ’s No e: MDPI s ays neu al wi h ega d o ju isdic ional claims in published maps and ins i u ional a il- ia ions. Copy igh : © 2022 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h ps:// c ea i ecommons.o g/licenses/by/ 4.0/). sepa a ions A icle T i-Reagen Homogena e Is a Sui able S a ing Ma e ial o UHPLC-MS Lipidomic Analysis Ola z F esnedo 1, Bea iz Abad-Ga cia 2and Yu i Rueda 1,* 1Lipids & Li e Resea ch G oup, Depa men o Physiology, Facul y o Medicine and Nu sing, Uni e si y o he Basque Coun y (UPV/EHU), 48940 Leioa, Spain 2 Cen al Analysis Se ice (SGIke ), Ma ina Casiano Pla o m, Uni e si y o he Basque Coun y (UPV/EHU), 48940 Leioa, Spain *Co espondence: yu i. [email p o ec ed] Abs ac : Backg ound: T ansc ip omic and lipidomic dual analyses usually ini ia e wi h independen ex ac i e p ocedu es. Tha en ails a di icul y in aligning esul s om bo h omics pla o ms, especially in he case o highly he e ogeneous issues, such as he kidney. Me hods: Bligh and Dye lipid ex ac ion was pe o med using a kidney homogena es p epa ed in PBS o comme cially a ailable T i- eagen used o RNA ex ac ion. Samples we e analyzed by ul ahigh pe o mance liquid ch oma og aphy-mass spec ome y (UHPLC-MS) lipidomic analysis. Resul s: Compa ison o he lipidome ob ained om phospha e-bu e ed saline (PBS) and T i- eagen homogena es showed quali a i e and quan i a i e alidi y o he T i- eagen homogena e wi h he excep ion o e he lipids; he acidic na u e o he mix seems o p omo e he hyd olysis o he e he bond, especially in plasmalogens. We es ed se e al condi ions in he sample p ocessing, which allowed o op imize he p ocedu e. Conclusions: Aiming o implemen a me hod ha allows he ex ac ion o RNA and lipids om he same issue homogena e no using ex e nal ace s, we he e epo he use o T i- eagen homogena es as a sui able s a ing ma e ial o UHPLC-MS lipidomic analysis. Keywo ds: lipidomic; kidney; lipid ex ac ion; RNA ex ac ion; T i- eagen ; UHPLC-MS 1. In oduc ion T ansc ip omic and me abolomic dual analysis in biological sys ems enables o in- eg a e he p ima y gene exp ession wi h pheno ypic esponses. Classic me hodologies ini ia e wi h independen ex ac i e p ocedu es in each omics wo k low. Rega ding RNA ex ac ion, he use o comme cially a ailable T i- eagen s based on he well-es ablished acid guanidinium hiocyana e-phenol-chlo o o m ex ac ion me hod [ 1 ] is one o he p e- e ed p o ocols. A ca e ul handling o samples usually ende s e y good quan i a i e and quali a i e eco e ies o o al RNA. The p ocedu e includes a s ep o phase sepa a- ion using chlo o o m. As desc ibed by Podecha d e al. [ 2 ], he disca ded hyd ophobic phase can be used as a sou ce o ma e ial o lipid ex ac ion in lipidomic analyses by gas ch oma og aphy. To assess he ep esen a i eness o he chlo o o mic phase, hey used a adioac i e a y acid as a s anda d [2]. Adding an ex e nal adioac i e label o samples adds unce ain y o he whole p o ocol and es ic s i s use o sui able acili ies. To a oid his ac o , in his wo k, we es ed he use o issue homogenized in T i- eagen , usually used o RNA ex ac ion, in a lipidomic anal- ysis by ul ahigh pe o mance liquid ch oma og aphy-mass spec ome y (UHPLC-MS). Compa ison o he esul s wi h a usual lipidomic analysis s a ing om issue homogenized in a s anda d bu e ed solu ion shows ha T i- eagen homogena e is a sui able s a ing ma e ial o UHPLC-MS lipidomic analyses. Al hough he acidic na u e o T i- eagen induces he hyd olysis o some e he -con aining lipids, we desc ibe ha keeping he ime be ween issue homogeniza ion and lipid ex ac ion sho and he ein o ced bu e ing o he ex ac ion mix u e sol es he p oblem. A common ex ac i e p ocedu e will be use ul Sepa a ions 2022,9, 268. h ps://doi.o g/10.3390/sepa a ions9100268 h ps://www.mdpi.com/jou nal/sepa a ions Sepa a ions 2022,9, 268 2 o 8 o mul i-omics app oaches in s udies o he e ogeneous issues/o gans, such as he kidney, whose complex his ological s uc u e is e lec ed in di e en ial lipidomes as i has been e ealed by imaging mass spec ome y o human and mouse samples [ 3 , 4 ]. In es iga ions on o he issues wi h he e ogeneous lipidome dis ibu ion, such as b ain [ 5 ] o cance ous issues, which a e cha ac e ized by in a- umo he e ogenei y [ 6 – 8 ], would bene i om he p o ocol desc ibed he e. 2. Ma e ials and Me hods 2.1. P epa a ion o Lipid Ex ac s To p epa e lipid ex ac s, kidneys we e ob ained om h ee emale Sp age-Dawley a s. Animal handling p ocedu es we e pe o med acco ding o he Uni e si y o he Basque Coun y e hical commi ee (M20/2016/237). Samples we e main ained in solid CO 2 un il homogeniza ion and kep a 0 ◦ C du ing homogeniza ion. A kidney om each animal was inely minced using a scalpel, and all issue agmen s we e pooled. Aliquo s o he issue pool we e homogenized in 10 olumes o phospha e-bu e ed saline (PBS; 10 mM phospha e bu e , pH 7.4, 150 mM NaCl) o T izol, he T i- eagen om In i ogen (Wal ham, MA, USA), using a Poly on homogenize (Kinema ica AG, Mal e s, Swi ze land) (12 mm dispe sing agg ega e, 1 min a 80% o maximum in ensi y). P o ein concen a ion was measu ed using he BCA assay (The mo Scien i ic, Wal ham, MA, USA). As he T izol eagen is incompa ible wi h he BCA assay, we used he p o ein concen a ion measu ed in PBS-homogena e as a e e ence. P o ein con ibu es o a ound 10% o esh kidney mass. In he i s expe imen s, homogena e olumes con aining 0.1, 0.3 and 0.5 mg o p o ein o PBS homogena es and he equi alen issue masses om T izol homogena es we e used o lipid ex ac ion. Homogena e olumes we e adjus ed o 600 µ L wi h PBS p io o lipid ex ac ion. In he las expe imen , he same p ocedu e was ollowed (only wi h 0.5 mg o p o ein), bu in some cases, 3 × concen a ed PBS was used o inc ease he bu e ing o he ex ac ion mix u e. In addi ion, in o de o obse e he e ec o ime be ween homogeniza ion and addi ion o homogena e o he lipid ex ac ion mix u e, ime was con olled and kep below 2 min o o e 5 min. Lipid ex ac ion is based on he p ocedu e desc ibed by Bligh and Dye [ 9 ]. B ie ly, 600 µ L o he sample we e added o 9 mL o chlo o o m:me hanol 1:2 ( : ). A his s ep, in e nal lipid s anda ds we e added o allow quan i ica ion in he lipidomic analysis: Splash LipidoMix, Ce amide/Sphingoid In e nal S anda d Mix u e I, Ca diolipin In e nal S anda d Mix u e, D18:1/12:0 monosul ogalac osyl ( β ) ce amide, 24:0 (d4) L-ca ni ine and oleic acid (d9), all om A an i Pola Lipids (Alabas e , AL, USA). The ollowing s eps we e pe o med a oom empe a u e: o ex mix (2 min), add 3 mL o chlo o o m, o ex mix (1 min), add 4.8 mL o wa e , o ex mix (1 min). Samples we e cen i uged (1200 × g, 15 min, 4 ◦ C) o phase sepa a ion. The lowe , chlo o o mic phase was collec ed, and he uppe , aqueous phase was e-ex ac ed a oom empe a u e: add 7.2 mL o chlo o o m:me hanol:wa e 1:1:1 ( : : ), o ex mix (2 min). Samples we e cen i uged as done p e iously, he chlo o o mic phase was collec ed and combined wi h he p e ious one. Chlo o o m was e apo a ed by cen i ugal e apo a ion (Sa an SpeedVac, The mo Elec on Company, Wal ham, MA, USA) and samples we e s o ed a − 80 ◦ C in a N 2 a mosphe e un il he lipidomic analysis. Chlo o o m and me hanol we e pu chased om Scha lau (Sen mena , Spain) and we e o ≥99.8% pu i y. 2.2. UHPLC-MS Analysis Lipidomic analysis was pe o med in he Cen al Analysis Se ice acili y o he Uni e si y o he Basque Coun y (SGIke UPV-EHU, Campus o Biscay, Leioa, Spain), and he p ocedu e was published p e iously [ 10 ]. Global lipidomic p o iles we e de e mined by andem MS using an elec osp ay ioniza ion sou ce (ESI) in nega i e ( − ) and posi i e mode (+) a e sepa a ion o lipid classes by a e e se-phase ul ahigh pe o mance liquid Sepa a ions 2022,9, 268 3 o 8 ch oma og aphy (UHPLC). The ch oma og aphic sepa a ion was achie ed on a Vanquish UHPLC sys em (The moFishe Scien i ic, Wal ham, MA, USA), equipped wi h a bina y sol en deli e y pump, a he mos a ed au osample and a column o en. A e e se-phase column (Acqui y UPLC C18 CSHTM 2.1 × 100 mm, 1.7 µ m) and a p e-column (Acqui y UPLC C18 CSHTM 2.1 mm × 5 mm, 1.7 µ m: VanGua d, Valley Fo ge, PA, USA), bo h pu chased om Wa e s (Mil o d, MA, USA), we e used a 65 ◦ C o sepa a e indi idual lipids. The mobile phases consis ed o ace oni ile and wa e (40:60, : ) wi h 10 mM ammonium o ma e and 0.1% o mic acid (phase A), and ace oni ile and isop opanol (10:90, : ) wi h 10 mM ammonium o ma e and 0.1% o mic acid (phase B). The applied elu ion condi ions we e: 0–2 min, 40–43% B; 2–2.1 min, 43–50% B; 2.1–12 min, 50–54% B, 12–12.1 min, 54–70% B; 12.1–18 min, 70–100% B. Finally, washing and econdi ioning o he column we e done. The low a e was 500 µ L/min, and he injec ion olume was 2 µ L. All samples we e kep a 10 ◦ C du ing he analysis. Op ima ® LC/MS-g ade wa e , me hanol, ace oni ile, isop opanol and o mic acid we e ob ained om Fishe Scien i ic (Wal ham, MA, USA). Ammonium o ma e was pu chased om Sigma-Ald ich (S . Louis, MO, USA). All UHPLC-MS/MS da a we e acqui ed on a Q Exac i e HF-X hyb id quad upole- O bi ap mass spec ome e (The moFishe Scien i ic, Wal ham, MA, USA) equipped wi h a HESI (hea ed elec osp ay ioniza ion) sou ce using a da a-dependend LC-MS/MS me hod ( op 15 MS2) in bo h posi i e mode and nega i e mode. The mass spec ome e se ings we e op imized using he Splash LipidoMix and Ce amide/Sphingoid In e nal S anda d Mix u e I (A an i Pola Lipids, Alabas e , AL, USA). The low a es o shea h gas, sweep gas and auxilia y gas o bo h pola i ies we e adjus ed o 35, 0 and 10 (a bi a y uni s). Fo bo h ioniza ion modes, he capilla y empe a u e and he hea e empe a u e we e main ained a 285 ◦ C and 370 ◦ C, espec i ely, while he sp ay ol age was 3.90 kV o posi i e and 3.20 kV o nega i e ioniza ion. The S-lens RF le el was se a 40. The O bi ap mass spec ome e was ope a ed a a esol ing powe o 120,000 in ull-scan mode (scan ange 250–2000 m/z, au oma ic gain con ol a ge 1 × 10 6 ) and 7500 in Top15 da a-dependen MS2 mode (HCD agmen a ion wi h a s epped no malized collision ene gy o 25 and 30 in posi i e mode, and 20, 30 and 40 in nega i e ion mode; injec ion ime 11 ms; isola ion window 1 m/z; au oma ic gain con ol a ge 1 × 10 5 wi h a dynamic exclusion se ing o 6.0 s). The spec ome e was calib a ed ex e nally e e y h ee days wi hin a mass accu acy o 1 ppm. 2.3. MS Da a P ocessing All he MS da a we e acqui ed and p ocessed using he Xcalibu so wa e package ( e sion 4.1, The mo Fishe Scien i ic, Wal ham, MA, USA), while he LipidSea ch so wa e e sion 4.2.27 (Mi sui Knowledge Indus y, Tokyo, Japan) was used o iden i y and quan i y he lipid species in hese complex biological samples. The key p ocessing pa ame e s we e as ollows: a ge da abase, Gene al; p ecu so ole ance, 5 ppm; p oduc ole ance, 5 ppm; p oduc ion h eshold, 1%; m-sco e h eshold, 2; Quan m/z ole ance, ± 5 ppm; Quan RT ( e en ion ime) ange, ± 0.5 min; use o main isome il e s and ID quali y il e s A, B, C and D; adduc ions H+, Na+ and NH4+ o posi i e ion mode, and H − and HCOO − o nega i e ion mode. A a ia ion coe icien h eshold o 30% was applied o he in ensi ies o masses assigned o lipid s uc u es om 10 uns o he quali y con ol mix o il e he da a acqui ed. Table 1summa izes he lipid classes de ec ed and he analyzed adduc s ( hose o highes in ensi ies in each lipid class). Sepa a ions 2022,9, 268 4 o 8 Table 1. Lipid classes de ec ed by UHPLC-MS a e lipid ex ac ion om PBS and T izol homogena es o kidney. Abb e ia ions (Abb .) and he adduc o he highes in ensi y a e indica ed o each lipid class. Lipid Ca ego y Lipid Class Abb . Mos In ense Adduc Glyce ophospholipid Phospha idylcholine PC [PC+H]+ E he -PC PCe [PC(O)+H]+ Lyso-PC LPC [LPC+HCOO]− Phospha idyle hanolamine PE [PE-H]− E he -PE PEe [PE(O/P)-H]− Lyso-PE LPE [LPE-H]− Phospha idylse ine PS [PS-H]− Phospha idylinosi ol PI [PI-H]− Phospha idylglyce ol PG [PG-H]− Ca diolipin CL [CL-H]− Sphingolipid Sphingomyelin SM [SM+H]− Hexosylce amide HexCe [HexCe +Na/H]+ Sul a ide ST [ST+HCOO]− Ce amide Ce [Ce +HCOO]− Neu al glyce olipid T iglyce ide TG [TG+NH4]+ Diglyce ide DG [DG+Na]+ 3. Resul s and Discussion This wo k aims o explo e whe he T i- eagen homogena es a e app op ia e o lipid ex ac ion. To do ha , we used T izol, T i- eagen manu ac u ed by In i ogen. In ou i s a emp o p epa e lipid ex ac s, we used he chlo o o mic phase om he T izol eagen p o ocol o RNA ex ac ion, as desc ibed p e iously [ 2 ]. To a oid he use o adioac i e ace s, we eco e ed he hyd ophobic phase (usually disca ded) in a s anda d RNA ex ac ion (50–100 mg issue in 10 olumes o eagen ). A e ex ac ion o lipids by he Bligh and Dye me hod and e apo a ion o chlo o o m, a non- ola ile esidue emained in he ex ac s ha made i unsui able o subsequen UHPLC-MS analysis. Tha is why we explo ed he possibili y o using he whole T izol homogena e o ex ac lipids. This allowed wo king wi h a homogeneous suspension and a ep esen a i e aliquo o lipid ex ac ion. To es ablish he alidi y o T izol homogena e as s a ing ma e ial o he de e mina ion o lipid composi ion o kidney, we pe o med a lipidomic analysis. Rep esen a i e base peak in ensi y ch oma og ams ob ained in ESI-posi i e and -nega i e modes o he UHPLC- MS analysis a e shown in Supplemen a y Figu e S1. F om he da a o he i s expe imen , we calcula ed he a e age pe cen age ha each lipid species’ in ensi y ep esen s in i s lipid class and compa ed he esul wi h ha om PBS-homogena e (Supplemen a y Figu e S2). Only classes wi h mo e han i e molecula species iden i ied we e conside ed o his analysis. The ne alues de ec ed in he ex ac s a e lis ed in Supplemen a y Table S1. Wi h he excep ion o lysoPE species, he lipid composi ion is almos iden ical in bo h ex ac s. Among lysoPEs, LPE(20:4) is he mos abundan species in he T izol ex ac , while LPE(16:0) and LPE(18:0) a e much lowe han in PBS ex ac s. Nex , o e alua e he quan i a i e esponse o he lipidomic analysis, we pe o med a linea i y analysis in lipid ex ac s om 0.1, 0.3 and 0.5 mg o p o ein. The objec i e was dual; on one hand, we wan ed o check whe he lipid de ec ion om T izol homogena es showed a good linea esponse agains p o ein quan i y. On he o he hand, we wan ed o ule ou a possible a i ac in LPE quan i ica ion. Lipidomic analysis esul s a e shown as he sum o in ensi ies o lipid species o each lipid class (Figu e 1); again, only classes wi h mo e han i e molecula species iden i ied we e conside ed o his analysis. Values a e plo ed agains he p o ein quan i y o kidney mass used o lipid ex ac ion; R 2 alues o each linea eg ession analysis a e shown. Sepa a ions 2022,9, 268 5 o 8 Sepa a ions 2022, 9, x FOR PEER REVIEW 5 o 8 a e plo ed agains he p o ein quan i y o kidney mass used o lipid ex ac ion; R2 alues o each linea eg ession analysis a e shown. Figu e 1. Lipid class in ensi y de ec ed by UHPLC-MS a e lipid ex ac ion om T izol o PBS ho- mogena es o kidney. (A) Values ep esen he sum o in ensi ies o lipid species o each lipid class; (B) alues co esponding o CL a e ep esen ed as in (A) and no malized wi h in ensi y o he in e - nal s anda d included be o e lipid ex ac ion (Ca diolipin Mix I om A an i Pola Lipids). Values a e plo ed agains he p o ein quan i y o kidney homogena es used o lipid ex ac ion. R2 alues o each linea eg ession analysis a e shown nex o each line. Only classes wi h mo e han 5 mo- lecula species iden i ied ha e been conside ed o his analysis. 2 (0.1 mg o p o ein) o 3 (0.3 and 0.5 mg o p o ein) homogena es we e used o lipid ex ac ion and da a a e shown as he mean ± SEM. The de ec ion o majo memb ane lipids (PC, PE, PI, PS, PG and SM—check Figu e 1 o meaning o abb e ia ions) and neu al lipids (Ce and TG) had good linea esponse agains p o ein quan i y and showed simila in ensi ies in he analysis o bo h homoge- na es (Figu e 1A). O e all, he beha io o summa ions o hose lipid classes e lec s ha o indi idual species in each class (Supplemen a y Table S1). In he case o CL, he esul s did no show a good linea esponse in bo h ex ac s (Figu e 1B), which indica es ha his unexpec ed esul is no a consequence o homogeniza ion in T izol. Al hough his seems o sugges ha he ex ac ion me hod is no quan i a i e o CL, when we no malized he in ensi y alues o he lipid species wi h hose o he in e nal s anda ds (Ca diolipin Mix I om A an i Pola Lipids) included be o e lipid ex ac ion, bo h homogena es showed good linea esponse and simila slopes. This emphasizes he necessi y o including lipid Figu e 1. Lipid class in ensi y de ec ed by UHPLC-MS a e lipid ex ac ion om T izol o PBS homogena es o kidney. ( A ) Values ep esen he sum o in ensi ies o lipid species o each lipid class; ( B ) alues co esponding o CL a e ep esen ed as in ( A ) and no malized wi h in ensi y o he in e nal s anda d included be o e lipid ex ac ion (Ca diolipin Mix I om A an i Pola Lipids). Values a e plo ed agains he p o ein quan i y o kidney homogena es used o lipid ex ac ion. R 2 alues o each linea eg ession analysis a e shown nex o each line. Only classes wi h mo e han 5 molecula species iden i ied ha e been conside ed o his analysis. 2 (0.1 mg o p o ein) o 3 (0.3 and 0.5 mg o p o ein) homogena es we e used o lipid ex ac ion and da a a e shown as he mean ±SEM. The de ec ion o majo memb ane lipids (PC, PE, PI, PS, PG and SM—check Figu e 1 o meaning o abb e ia ions) and neu al lipids (Ce and TG) had good linea esponse agains p o ein quan i y and showed simila in ensi ies in he analysis o bo h homogena es (Figu e 1A). O e all, he beha io o summa ions o hose lipid classes e lec s ha o indi idual species in each class (Supplemen a y Table S1). In he case o CL, he esul s did no show a good linea esponse in bo h ex ac s (Figu e 1B), which indica es ha his unexpec ed esul is no a consequence o homogeniza ion in T izol. Al hough his seems o sugges ha he ex ac ion me hod is no quan i a i e o CL, when we no malized he in ensi y alues o he lipid species wi h hose o he in e nal s anda ds (Ca diolipin Mix I om A an i Pola Lipids) included be o e lipid ex ac ion, bo h homogena es showed good linea esponse and simila slopes. This emphasizes he necessi y o including lipid s anda ds in ex ac ion p o ocols no only because i allows o quan i ica ion, bu also because i can co ec p ocedu e e o s in mino i y lipids. In he case o PCe and lysophospholipids, he slopes a e subs an ially dis inc be ween PBS and T izol homogena es, al hough he R 2 o he linea eg ession is good in gene al. In Sepa a ions 2022,9, 268 6 o 8 he case o PEe, no only we e slopes subs an ially dis inc , bu in addi ion, T izol samples did no show a cohe en esponse agains p o ein quan i y. We canno exclude he possibili y ha du ing sample p ocessing some PE and PEe may deg ade and p oduce lysoPE. A achidonic acid (20:4) is he mos abundan a y acid in he sn-2 posi ion o he glyce ol backbone o PE and PEe species; palmi ic acid (16:0) and s ea ic acid (18:0) occupy he sn-1 posi ion in mo e han 60% o PE. Small di e ences in suscep ibili y o hyd olysis du ing sample p ocessing in PBS and T izol (which is acidic) migh accoun o he di e ences in LPE composi ion. In ac , plasmalogens (mos o he PEe species in ESI-posi i e da a; no shown) a e mo e sensi i e o acidic hyd olysis [ 11 ], which is a oided in he bu e ed PBS homogena es. To e i y i acidic hyd olysis was esponsible o he a o emen ioned di e ences, we epea ed he lipid ex ac ion and lipidomic analysis o samples equi alen o 0.5 mg o p o ein. In his case, sample homogeniza ion was ca ied ou as p e iously (in PBS o T izol), bu also in T izol wi h s ic con ol o he ime ( ≤ 2 min) be ween homogeniza ion and he ini ia ion o he lipid ex ac ion p ocedu e, and in some cases wi h ex a bu e ing wi h PBS in he lipid ex ac ion. A e wa ds, lipid ex ac ion, lipidomic analysis and da a p ocessing we e pe o med as done p e iously. Figu e 2shows p opo ions be ween e he -con aining majo phospholipids and hei co esponden lysophospholipids. T izol homogena es showed a d ama ic dec ease in PEe/LPE p opo ion when he ime be ween homogeniza ion and lipid ex ac ion was o e 5 min and no ex a bu e ing was in oduced. This is in acco dance wi h esul s shown in Figu e 1and Figu e S1. When ime was dec eased o ≤ 2 min PEe/LPE p opo ion was es o ed and ex a bu e ing wi h mo e concen a ed PBS imp o ed he p opo ion in bo h lipids o alues abo e hose obse ed in he con ol. Sepa a ions 2022, 9, x FOR PEER REVIEW 6 o 8 s anda ds in ex ac ion p o ocols no only because i allows o quan i ica ion, bu also because i can co ec p ocedu e e o s in mino i y lipids. In he case o PCe and lysophospholipids, he slopes a e subs an ially dis inc be- ween PBS and T izol homogena es, al hough he R2 o he linea eg ession is good in gene al. In he case o PEe, no only we e slopes subs an ially dis inc , bu in addi ion, T izol samples did no show a cohe en esponse agains p o ein quan i y. We canno exclude he possibili y ha du ing sample p ocessing some PE and PEe may deg ade and p oduce lysoPE. A achidonic acid (20:4) is he mos abundan a y acid in he sn-2 posi ion o he glyce ol backbone o PE and PEe species; palmi ic acid (16:0) and s ea ic acid (18:0) occupy he sn-1 posi ion in mo e han 60% o PE. Small di e ences in suscep ibili y o hyd olysis du ing sample p ocessing in PBS and T izol (which is acidic) migh accoun o he di e ences in LPE composi ion. In ac , plasmalogens (mos o he PEe species in ESI-posi i e da a; no shown) a e mo e sensi i e o acidic hyd olysis [11], which is a oided in he bu e ed PBS homogena es. To e i y i acidic hyd olysis was esponsible o he a o emen ioned di e ences, we epea ed he lipid ex ac ion and lipidomic analysis o samples equi alen o 0.5 mg o p o ein. In his case, sample homogeniza ion was ca ied ou as p e iously (in PBS o T i- zol), bu also in T izol wi h s ic con ol o he ime (≤2 min) be ween homogeniza ion and he ini ia ion o he lipid ex ac ion p ocedu e, and in some cases wi h ex a bu e ing wi h PBS in he lipid ex ac ion. A e wa ds, lipid ex ac ion, lipidomic analysis and da a p ocessing we e pe o med as done p e iously. Figu e 2 shows p opo ions be ween e he -con aining majo phospholipids and hei co esponden lysophospholipids. T izol homogena es showed a d ama ic dec ease in PEe/LPE p opo ion when he ime be ween homogeniza ion and lipid ex ac ion was o e 5 min and no ex a bu e ing was in oduced. This is in acco dance wi h esul s shown in Figu es 1 and S1. When ime was dec eased o ≤2 min PEe/LPE p opo ion was es o ed and ex a bu e ing wi h mo e concen a ed PBS imp o ed he p opo ion in bo h lipids o alues abo e hose obse ed in he con ol. Figu e 2. P opo ions be ween majo e he -con aining phospholipids and hei lyso-de i a i es. Ho- mogeniza ion was pe o med in PBS (P) o T izol (T), ime be ween lipid ex ac ion and homogeni- za ion was kep unde 2 min o no and bu e ing o he lipid ex ac ion mix u e wi h PBS was a iable (see Ma e ials and Me hods sec ion). Da a co espond o n = 5 and a e shown as he mean ± SEM. S uden ’s - es : * p < 0.05; ** p < 0.01; *** p < 0.001. In summa y, his wo k shows ha he kidney T i- eagen homogena e is a sui able s a ing ma e ial o lipid ex ac ion and lipidomic analysis by UHPLC-MS. An adequa e p ocedu e, in e ms o ime con ol and bu e ing o samples in homogeniza ion and lipid ex ac ion, leads o lipidomics esul s compa able o hose ob ained wi h samples homog- enized in a usual bu e , such as PBS. Aiming o con ibu e an easy- o- ollow guide ha includes all men ioned s eps, we p o ide a e ised p o ocol o adequa e sample p o- cessing and lipid ex ac ion (Figu e 3). This p ocedu e does no hinde any o he s eps o Figu e 2. P opo ions be ween majo e he -con aining phospholipids and hei lyso-de i a i es. Homogeniza ion was pe o med in PBS (P) o T izol (T), ime be ween lipid ex ac ion and homog- eniza ion was kep unde 2 min o no and bu e ing o he lipid ex ac ion mix u e wi h PBS was a iable (see Ma e ials and Me hods sec ion). Da a co espond o n= 5 and a e shown as he mean ± SEM. S uden ’s - es : * p< 0.05; ** p< 0.01; *** p< 0.001. In summa y, his wo k shows ha he kidney T i- eagen homogena e is a sui able s a ing ma e ial o lipid ex ac ion and lipidomic analysis by UHPLC-MS. An adequa e p ocedu e, in e ms o ime con ol and bu e ing o samples in homogeniza ion and lipid ex ac ion, leads o lipidomics esul s compa able o hose ob ained wi h samples homogenized in a usual bu e , such as PBS. Aiming o con ibu e an easy- o- ollow guide ha includes all men ioned s eps, we p o ide a e ised p o ocol o adequa e sample p ocessing and lipid ex ac ion (Figu e 3). This p ocedu e does no hinde any o he s eps o he RNA ex ac ion p o ocol, as i only en ails sha ing he issue sample o lipid and RNA ex ac ion. Using he same s a ing ma e ial o bo h ansc ip omic and lipidomic analyses will imp o e he alignmen o esul s om bo h omics pla o ms. This will be use ul, especially in he case o highly he e ogeneous issues, such as he kidney, because Sepa a ions 2022,9, 268 7 o 8 e en adjacen small agmen s can ha e di e en pheno ypes, including lipidome and ansc ip ome [3,4,12]. Sepa a ions 2022, 9, x FOR PEER REVIEW 7 o 8 he RNA ex ac ion p o ocol, as i only en ails sha ing he issue sample o lipid and RNA ex ac ion. Using he same s a ing ma e ial o bo h ansc ip omic and lipidomic anal- yses will imp o e he alignmen o esul s om bo h omics pla o ms. This will be use ul, especially in he case o highly he e ogeneous issues, such as he kidney, because e en adjacen small agmen s can ha e di e en pheno ypes, including lipidome and an- sc ip ome [3,4,12]. Figu e 3. Op imized p o ocol o sample p ocessing and lipid ex ac ion s a ing om a issue piece aken o RNA ex ac ion wi h T i- eagen [9]. Supplemen a y Ma e ials: The ollowing suppo ing in o ma ion can be downloaded a : www.mdpi.com/xxx/s1, Figu e S1: Rep esen a i e base peak in ensi y ch oma og ams o PBS- and T izol-homogenized samples analyzed by UHPLC-MS. Figu e S2: Dis ibu ion o lipid species among each lipid class; Table S1: In ensi y o all lipid species iden i ied in lipidomic analysis. Au ho Con ibu ions: Concep ualiza ion, O.F. and Y.R.; Fo mal analysis, O.F., B.A.-G. and Y.R.; Funding acquisi ion, O.F. and Y.R.; Me hodology, O.F. and Y.R.; W i ing—o iginal d a , O.F. and Y.R.; W i ing— e iew and edi ing, O.F., B.A.-G. and Y.R. All au ho s ha e ead and ag eed o he published e sion o he manusc ip . Figu e 3. Op imized p o ocol o sample p ocessing and lipid ex ac ion s a ing om a issue piece aken o RNA ex ac ion wi h T i- eagen [9]. Supplemen a y Ma e ials: The ollowing suppo ing in o ma ion can be downloaded a : h ps: //www.mdpi.com/a icle/10.3390/sepa a ions9100268/s1, Figu e S1: Rep esen a i e base peak in- ensi y ch oma og ams o PBS- and T izol-homogenized samples analyzed by UHPLC-MS. Figu e S2: Dis ibu ion o lipid species among each lipid class; Table S1: In ensi y o all lipid species iden i ied in lipidomic analysis. Au ho Con ibu ions: Concep ualiza ion, O.F. and Y.R.; Fo mal analysis, O.F., B.A.-G. and Y.R.; Funding acquisi ion, O.F. and Y.R.; Me hodology, O.F. and Y.R.; W i ing—o iginal d a , O.F. and Y.R.; W i ing— e iew and edi ing, O.F., B.A.-G. and Y.R. All au ho s ha e ead and ag eed o he published e sion o he manusc ip . Funding: This esea ch was unded by he Basque Go e nmen (g an s IT971-16, IT1476-22 and KK-2020/00069) and he Spanish Minis y o Science and Inno a ion (PID2021-124425OB-100). Sepa a ions 2022,9, 268 8 o 8 Ins i u ional Re iew Boa d S a emen : The s udy was conduc ed in acco dance wi h he Decla a ion o Helsinki, and app o ed by E hics Commi ee on Animal Expe imen a ion o he Uni e si y o he Basque Coun y (p o ocol code: M20/2016/237 and da e o app o al: 2 Decembe 2016). In o med Consen S a emen : No applicable. Da a A ailabili y S a emen : No applicable. Con lic s o In e es : The au ho s decla e no con lic o in e es . The unde s had no ole in he design o he s udy; in he collec ion, analyses, o in e p e a ion o da a; in he w i ing o he manusc ip ; o in he decision o publish he esul s. Re e ences 1. Chomczynski, P.; Sacchi, N. Single-s ep me hod o RNA isola ion by acid guanidinium hiocyana e-phenol-chlo o o m ex ac ion. Anal. Biochem. 1987,162, 156–159. [C ossRe ] 2. Podecha d, N.; Ducheix, S.; Polizzi, A.; Lasse e, F.; Mon agne , A.; Legagneux, V.; Fouche, E.; Saez, F.; Lobacca o, J.M.; Lakhal, L.; e al. 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