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. Dual ex ac ion o mRNA and lipids om a single biological sample. Sci. Rep. 2018,8, 7019. [C ossRe ] [PubMed]
3.
Ma ín-Saiz, L.; Mos ei o, L.; Solano-I u i, J.D.; Rueda, Y.; Ma ín-Allende, J.; Imaz, I.; Olano, I.; Ochoa, B.; F esnedo, O.;
Fe nández, J.A.; e al. High- esolu ion human kidney molecula his ology by imaging mass spec ome y o lipids. Anal. Chem.
2021,93, 9364–9372. [C ossRe ] [PubMed]
4.
Ma ín-Saiz, L.; Gue e o-Mau ecin, J.; Ma ín-Sanchez, D.; F esnedo, O.; Gómez, M.J.; Ca asco, S.; Canna a-O iz, P.; O iz,
A.; Fe nández, J.A.; Sanz, A.B. Fe os a in-1 modula es dys egula ed kidney lipids in acu e kidney inju y. J. Pa hol.
2022
,257,
285–299. [C ossRe ] [PubMed]
5.
Veloso, A.; As iga aga, E.; Ba eda-Gómez, G.; Manuel, I.; Fe e , I.; Gi al , M.T.; Ochoa, B.; F esnedo, O.; Rod íguez-Pue as, R.;
Fe nández, J.A. Ana omical dis ibu ion o lipids in human b ain co ex by imaging mass spec ome y. J. Am. Soc. Mass Spec om.
2011,22, 329–338. [C ossRe ] [PubMed]
6. McG anahan, N.; Swan on, C. Clonal he e ogenei y and umo e olu ion: Pas , p esen , and he u u e. Cell 2017,168, 618–628.
7.
Te y, S.; Engelsen, A.S.T.; Bua , S.; Elsayed, W.S.; Venka esh, G.H.; Chouaib, S. Hypoxia-d i en in a umo he e ogenei y and
immune e asion. Cance Le . 2020,492, 1–10. [C ossRe ] [PubMed]
8.
Biswas, A.; De, S. D i e s o dynamic in a umo he e ogenei y and pheno ypic plas ici y. Am. J. Physiol. Cell Physiol.
2021
,320,
C750–C760. [C ossRe ] [PubMed]
9.
Bligh, E.G.; Dye , W.J. A apid me hod o o al lipid ex ac ion and pu i ica ion. Can. J. Biochem. Physiol.
1959
,37, 911–917.
[C ossRe ] [PubMed]
10.
I iondo, A.; Tain a, M.; Saldias, J.; A iba, M.; Ochoa, B.; Goni, F.M.; Ma inez-Lage, B.; Abad-Ga cia, B. Isop opanol ex ac ion o
ce eb ospinal luid lipidomic p o iling analysis. Talan a 2019,195, 619–627. [C ossRe ] [PubMed]
11.
F osolono, M.F.; Rappo , M.M. Reac i i y o plasmalogens: Kine ics o acid-ca alyzed hyd olysis. J. Lipid Res.
1969
,10, 504–506.
[C ossRe ]
12.
Lee, J.W.; Chou, C.L.; Kneppe , M.A. Deep sequencing in mic odissec ed enal ubules iden i ies neph on segmen -speci ic
ansc ip omes. J. Am. Soc. Neph ol. 2015,26, 2669–2677. [C ossRe ] [PubMed]
