Nonionic Su ac an s can Modi y he The mal S abili y o Globula
and Memb ane P o eins In e e ing wi h he The mal P o eome
P o iling P inciples o Iden i y P o ein Ta ge s
Emmanuel Be lin,
⊥
Ve onica Lizano-Fallas,
⊥
Ana Ca asco del Amo , Ola z F esnedo,
and Susana C is obal*
Ci e This: Anal. Chem. 2023, 95, 4033−4042
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sı Suppo ing In o ma ion
ABSTRACT: The memb ane p o eins a e essen ial a ge s o unde s anding
cellula unc ion. The unbiased iden i ica ion o memb ane p o ein a ge s is s ill he
bo leneck o a sys em-le el unde s anding o cellula esponse o s imuli o
pe u ba ions. I has been sugges ed o en ich he soluble p o eome wi h memb ane
p o eins by in oducing nonionic su ac an s in he solubiliza ion solu ion. This
s a egy aimed o simul aneously iden i y he globula and memb ane p o ein a ge s
by he mal p o eome p o iling p inciples. Howe e , he he mal shi assay would
su pass he cloud poin empe a u e om he nonionic su ac an s equen ly
u ilized o memb ane p o ein solubiliza ion. I is expec ed ha a ound he cloud
poin empe a u e, he su ac an micelles would su e s uc u al modi ica ions
al e ing p o ein solubili y. He e, we show ha he p esence o nonionic su ac an s
can al e p o ein he mal s abili y om a mixed, globula , and memb ane p o eome.
In he p esence o su ac an micelles, he changes in p o ein solubili y analyzed a e
he he mal shi assay was a ec ed by he he mally dependen modi ica ion o he micella size and i s in e ac ion wi h p o eins. We
demons a e ha he in oduc ion o nonionic su ac an s o he solubiliza ion o memb ane p o eins is no compa ible wi h he
p inciples o a ge iden i ica ion by he mal p o eome p o iling me hodologies. Ou esul s lead o explo ing he mally independen
s a egies o memb ane p o ein solubiliza ion o assu e con iden memb ane p o ein a ge iden i ica ion. The p o eome-wide
he mal shi me hods ha e al eady shown hei capabili y o elucida e mechanisms o ac ion om pha ma, biomedicine, analy ical
chemis y, o oxicology, and inding s a egies, ee om su ac an s, o iden i y memb ane p o ein a ge s would be he nex
challenge.
■INTRODUCTION
Unde s anding he consequence o he in e ac ion o
memb ane p o eins wi h small molecules such as d ugs o
chemicals would equi e high- h oughpu me hodologies
suppo ing a apid sc eening o se e al housand p o ein
candida es om any cell ype. The memb ane p o eins a e
essen ial a ge s o unde s anding cellula unc ion. They a e
key nodes ha egula e cell communica ion and ini ia ion o
signal ansduc ion pa hways. Memb ane p o eins a e la gely
ep esen ed among he d ug a ge s o hei capabili y o
o ches a e cellula esponses.
1
Mo eo e , he memb ane
p o eins a e p ima y in e ac o s wi h small chemicals p esen
in ex acellula compa men s, could acili a e he cellula
up ake,
2
and could be esponsible o he molecula ini ia ion
e en s leading o ad e se ou come pa hways o human
heal h.
3
Di e en me hodologies ha e been applied o memb ane
p o ein a ge iden i ica ion. In gene al, he e we e me hods
wi h low h oughpu capabili y and a su icien simila i y o
p e iously desc ibed a ge s as a p e equisi e o new indings.
1
Impo an changes in he ield o a ge engagemen a ise wi h
he in oduc ion o he p o ein he mal shi assay in a
p o eome-wide con ex . The Cellula The mal Shi Assay
elayed on a la ge collec ion o an ibodies o p o ein
iden i ica ion and quan i a ion.
4
Howe e , he in oduc ion
o mass spec ome y (MS) o analyze he he mally induced
changes o p o eome solubili y was pi o al o enable he
unbiased iden i ica ion o a ge s om soluble p o eome in
me hods called The mal P o eome P o iling (TPP)
5
and la e
P o eome In eg al Solubili y Al e a ion Assay.
5,6
The de elop-
men s o hese me hodological app oaches ha e ecen ly
explo ed many scien i ic p oblems de ined by in e ac ions o
small chemicals and cellula p o eins om biomedicine and
analy ical chemis y.
7−10
A ou lab, we ha e ocused on
Recei ed: Oc obe 12, 2022
Accep ed: Janua y 24, 2023
Published: Feb ua y 13, 2023
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implemen a ions o i s applica ion o biodisco e y
11
o
oxicology.
12
Sea ching o a ge s in p o eomes con aining globula
p o eins acili a es he applica ion o his me hodology because
he p o ein−chemical in e ac ion is he main o only s imulus
ha induces pe u ba ion in p o ein solubili y unde he
he mal ea men . The ac o s a ec ing and uling p o ein
solubili y enla ge when he s udied p o eome con ains a
mix u e o globula and memb ane p o eins. The e o e, he
nex challenge was how o implemen hese me hods o he
iden i ica ion o memb ane p o ein a ge s. The epo ed
s a egy aims o enla ge he soluble p o eome wi h he addi ion
o nonionic su ac an s o he solubiliza ion bu e . The
su ac an s es ed a e ou inely used in memb ane p o ein
s udies, such as nonyl-phenyl-poly(e hylene glycol) (NP-
40S)
13
o Igepal CA-630 (Igepal), composed o oc yl-
phenoxy(polyoxye hylene)e hanol.
14
These nonionic su ac an s a e subs i u es o he o iginal
oc yl-phenoxy(polyoxye hylene)e hanol (NP-40) ha ,
al hough i has been widely used, is no longe p oduced.
The chemical s uc u e o Igepal esembles he o iginal NP-40
o mula ion be e han he new NP-40S. Howe e , NP-40S
o e s close alues o NP-40 in pa ame e s ha a e impo an
o micelle o ma ion, such as hyd ophile−lipophile balance o
molecula weigh .
15
The cloud poin empe a u e (CPT) is
one o he mos dis inc physical p ope ies o nonionic
su ac an s. I is he empe a u e abo e which a micella
solu ion o nonionic su ac an spon aneously o ms a wo-
phase sepa a ion. The aqueous micella wo-phase sys em has
been equen ly used o he ac iona ion o p o eins wi h
di e en hyd ophobici y.
16
Fo NP-40 and NP-40S, he CPT
occu s a 45−50 and 53−67 °C o Igepal. Looking in o he
TPP wo k low o he iden i ica ion o memb ane p o eins, i is
impo an o obse e ha he he mal shi spans om 37 o 67
°C. This empe a u e ange includes he speci ic CPTs o he
nonionic su ac an s used o inc ease he memb ane p o ein
solubiliza ion.
13
The expec ed empe a u e-dependen modi ica ions o he
solu ion wi h micelles o nonionic su ac an s would include a
leas micella s a i ica ion.
17
Addi ionally, du ing he inc eased
empe a u e and achie emen o CPT, he su ac an micelles
a e dehyd a ed, acqui ing an enla ged and elonga ed shape.
18
O e he CPT, i was epo ed ha micelle-embedded p o eins
would be o e sa u a ed in he micelle- ich laye while non-
micelle-bound p o eins exis in he aqueous- ich laye .
19
This
may cause c owding, p o ein−p o ein in e ac ions, micelle−
p o ein in e ac ions, micelle agg ega ion, and di e se changes
in he local en i onmen o he p o eome, which may a ec
p o ein s abili y.
20,21
Gi en he nume ous s udies based on he solubiliza ion o
memb ane p o eins wi h nonionic su ac an s, i has been
assumed ha he memb ane p o eome in micelles o nonionic
su ac an s could be compa ible wi h he TPP p inciples.
13
The
TPP p inciples o he iden i ica ion o p o ein a ge s a e
solely based on unique al e a ions in p o ein solubili y,
speci ically, he bene icial e ec s o p o ein−chemical in e -
ac ions o inc ease p o ein he mal ole ance.
5
The e o e,
insu icien a en ion has been paid o e alua ing he
modi ica ion o he aqueous micella solu ion a a empe a u e
close o o o e su ac an CPT and i he s uc u al and
densi y changes on he sol en solu ion could con ibu e o
al e ing he p o ein s abili y in solu ion independen ly o any
p o ein−chemical in e ac ion. This e alua ion would o e new
insigh s o de ine i he chemical−p o ein in e ac ion in
solubili y is s ill a obus pa ame e o iden i y memb ane
p o ein a ge s in a solu ion con aining nonionic su ac an
micelles ha a e e y malleable wi hin he he mal shi ange
o empe a u es.
In his s udy, we e alua e he dynamics o nonionic
su ac an micelles along wi h he he mal shi me hodology
and e alua e he empe a u e-dependen al e a ion o he
solubili y o he soluble and memb ane p o eome. The
in e ac ions o nonionic su ac an s wi h he soluble and
memb ane p o eome could al e hei he mal s abili y
in e e ing wi h he p o eome-wide he mal shi p inciples.
Conside ing he u gency o o e high- h ough me hodologies
o he unbiased iden i ica ion o memb ane p o ein a ge s,
his s udy aims o p o ide he ac o s and cons ain s o he
u u e implemen a ion o TPP o he iden i ica ion o
memb ane p o eins.
■MATERIALS AND METHODS
Collec ion o Li e Tissue. Li e s om 2-mon h-old
emale Sp ague Dawley a s we e ob ained om he Uni e si y
o he Basque Coun y wi h an e hics app o al code o M20/
2016/237. The p ocedu es conduc ed on he animals we e
app o ed by he E hics Commi ee o Animal Wel a e o he
Uni e si y o he Basque Coun y UPV/EHU and ollowed he
EU Di ec i es o animal expe imen a ion.
Nonionic Su ac an s in he S udy. Oc yl-phenoxy-
(polyoxye hylene)e hanol, named Igepal CA-630 (Igepal), and
nonyl-phenyl-poly(e hylene glycol), named Nonide -P40 sub-
s i u e (NP-40S).
Soluble P o eome. Li e issue was esuspended in bu e
con aining PBS (con ol), 0.4% ( / ) Igepal, o 0.4% ( / )
NP-40S in PBS and solubilized.
13
The insoluble ac ion was
sedimen ed by cen i uga ion a 100,000g o 60 min a 4 °C.
11
The soluble p o eome was used o pe o m he he mal
p o eome p o iling assay. The pelle s we e lysed wi h 100 μL o
RIPA bu e (RIPA Lysis Bu e , BOSTER) o pe o m p o ein
iden i ica ion and quan i ica ion o he insoluble ac ion.
P o ein concen a ion was de e mined by he BCA assay.
22
The mal Shi Assay (TSA). The soluble ac ions o he
h ee condi ions (con ol, Igepal, and NP-40S) we e hea ed by
duplica e a he speci ic en empe a u es selec ed o he
he mal shi assay: 37, 42, 46, 49, 51, 53, 55, 58, 62, and 67
°C. Aliquo s con aining 50 μg o p o ein we e independen ly
hea ed a he co esponding empe a u e o 3 min, ollowed
by 3 min a oom empe a u e using a he mocycle (MJ Mini
Pe sonal The mal Cycle PTC 1148, Bio-Rad). Samples we e
cen i uga ed a 100,000g o 20 min a 4 °C.
Mass Spec ome y. The samples we e econs i u ed wi h
0.1% o mic acid in ul a-pu e milli-Q wa e and sepa a ed
using an EASY nLC 1200 sys em (The mo Scien i ic).
Pep ides we e injec ed in o a p ecolumn (Acclaim PepMap
100 Å, 75 μm×2 cm) and sepa a ed on an EASY-Sp ay C18
e e sed-phase nano LC column (PepMap RSLC C18, 2 μm,
100 Å, 75 μm×25 cm). This was applied o a pe iod o 78
min, ollowed by 40% bu e B agains bu e A o 95 min;
ch oma og aphic g adien s and MS se ings a e a ailable in he
Suppo ing In o ma ion File.
Pep ide and P o ein Iden i ica ion and Quan i ica-
ion. The p o eins we e iden i ied using P o eome Disco e e
( e sion 2.1, The mo Fishe Scien i ic). The MS/MS spec a
( aw iles) we e sea ched by Seques HT agains heRa us
no egicus da abase om UniP o (UP000002494; 47,954
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en ies). A maximum o 2 yp ic clea ages we e allowed, and
he p ecu so and agmen mass ole ance we e 10 ppm and
0.02 Da, espec i ely. Pep ides wi h a alse disco e y a e
(FDR) o less han 0.01 and alida ion based on he q- alue
we e used as iden i ied. The minimum pep ide leng h
conside ed was 6, and he FDR was se o 0.1. P o eins we e
quan i ied using he a e age o he op h ee pep ide MS1
a eas, yielding aw p o ein abundances. Common con ami-
nan s like human ke a in and bo ine ypsin we e also included
in he da abase du ing he sea ches o minimize alse
iden i ica ions. The mass spec ome y p o eomics da a ha e
been deposi ed o he P o eomeXchange Conso ium ia he
PRIDE pa ne eposi o y wi h he da ase iden i ie
PXD037153.
23
P o ein Localiza ion. The bioin o ma ics ool PAN-
THER,
24
which u ilizes Gene On ology classi ica ion, was
used o p o ein localiza ion. No e ha one p o ein may ha e
se e al sublocaliza ions. This selec ion was made by choosing
p o eins agged wi h he GO e m: GO:0016020.
Dynamic Ligh Sca e ing (DLS). Solu ions we e PBS as
con ol, 0.4% Igepal, and 0.4% NP-40S. The empe a u es
chosen we e hose assayed in he TSA: 37, 42, 46, 49, 51, 53,
Figu e 1. Analysis o he dis ibu ion o memb ane p o eins in he soluble ac ions a e solubiliza ion wi h di e en ex ac ion solu ions. (A) Ba
diag am ep esen ing he dis ibu ion o he memb ane p o eins in he soluble ac ion based on GO ca ego ies and (B) ba diag am ep esen ing
changes in he dis ibu ion o memb ane p o eins in di e en subcellula loca ions. The ex ac ion solu ions we e con ol PBS, 0.4% ( / ) Igepal in
PBS, and 0.4% ( / ) NP-40S in PBS. The ac ions we e analyzed by quan i a i e mass spec ome y (nLC-MS/MS). Da a we e classi ied ia
PANTHER,
24
ollowing gene on ology classi ica ion.
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55, 58, 62, and 67 °C. To educe condensa ion, a silicon
solu ion was added on op o each well. The DLS (DynP o II
Pla e eade , Wya ) was se o inc ease he pla e empe a u e
ollowing he TSA p o ocol, and o each empe a u e, 10
acquisi ion imes (s) we e pe o med. Images we e aken
be ween he empe a u es 37 and 49 °C since empe a u es
abo e we e oo high o he came a o ope a e sa ely. Du ing
his he mal exposu e, bo h eadings o micelle size and images
we e aken. Ano he DLS expe imen was pe o med o
measu e changes in he DLS measu emen s om samples
hea ed a 55 and 67 °C (CPTs) and hen cooled o 21 °C, like
he TPP me hod ha includes he cooling s ep a RT o 3 min
be o e ul acen i uga ion. All expe imen s we e pe o med in
iplica e.
Figu e 2. Hea map ep esen ing he e ec o he empe a u e on he al e a ion o p o ein solubili y in aqueous micella solu ions. The soluble
ac ions we e sepa a ely exposed o a ange o empe a u es as in TPP me hodologies. The ac ions we e analyzed by quan i a i e mass
spec ome y (LC-MS/MS). The p o eins we e classi ied by (A) globula o (B) memb ane p o ein ia UniP o using Gene On ology classi ica ion.
The x-axis ep esen s he empe a u es, he y-axis ep esen s p o eins, and each ow is one p o ein. P o eins we e no malized agains he lowes
empe a u e (37 °C). Each alue is he mean o wo eplica es.
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Mic oscopy. Visualiza ion o samples exposed o hea was
pe o med o de ec su ac an and/o p o ein agg ega es by
p epa ing he samples ollowing he TPP me hod. Con ol,
Igepal, and NP-40S condi ions in a olume o 210 μL each
we e subjec ed o 67 °C o 3 min in a he mocycle . This
empe a u e was se o ensu e ha bo h, Igepal and NP-40S,
samples ha e passed hei CPT. A e 3 min o hea ing, 200 μL
o each sample was pu in o a glass bo om pla e (P35-G-1.5-
10-C, Ma Tek), and a co e glass was laid on op o he well. A
Leica DMi9 mic oscope was used wi h a moun ed hea ing box
se a 51 °C (maximum empe a u e o he hea ing box) o
slow down he cooling o he sample. Visualiza ion was
pe o med wi hin he i s 3 min using an HC PL APO CS2
63x/1.20 WATER UV objec i e wi h a TL-DIC con as o 10
ms exposu e o each image.
■RESULTS AND DISCUSSION
Solubiliza ion o Memb ane P o eins o TPP Anal-
ysis. The in oduc ion o nonionic su ac an s, Igepal o NP-
40S,
13
in he ex ac ion solu ion aims o inc ease he
iden i ica ion o memb ane p o ein a ge s om a p o eome
in a na i e s a e as is equi ed by he TPP me hodologies. We
e alua ed he composi ion and dis ibu ion o p o ein classes in
he soluble p o eome ex ac ed unde na i e condi ions wi h an
aqueous solu ion o PBS con aining a nonionic su ac an
Igepal o NP-40S in compa ison o he PBS-ex ac ed
p o eome as a con ol. He e, we analyzed by quan i a i e
p o eomics he soluble p o eome, a sample ha could be
u he u ilized o TPP, and he insoluble ac ion collec ed in
he pelle a e cen i uga ion. The numbe o p o eins in each
hi was no malized agains he o al numbe o p o eins. The
numbe o memb ane p o eins solubilized in he di e en
solu ions we e simila , 23% in PBS (1,550 p o eins), 24% in
Igepal (1,618 p o eins), and 22% in NP-40S (1,403 p o eins).
Howe e , looking a speci ic GO ca ego ies ha con ain
in eg al memb ane p o eins, such as he lea le o memb ane
bilaye and he side o memb ane, NP-40S inc eased by 50%
and Igepal by 20% in he numbe o hese memb ane p o eins
in he soluble ac ion compa ed o con ol (Figu e 1A,B).
The ex ac ion me hods ba ely inc eased he di e si y o
memb ane p o eins in he solu ion, al hough nonionic
su ac an s abo e hei c i ical micella concen a ion (CMC)
we e in oduced in he ex ac ion solu ion. Simila solubiliza-
ion solu ions o TPP analysis wi h samples om K562 cells
ende ed only 18% o memb ane p o eins in he solu ion.
13
The condi ions o acili a e ha a memb ane p o ein would
embed in a micelle a e complex and equi e he con e gence o
many mo e ac o s in addi ion o he su ac an concen a ion
in he ex ac ion solu ion.
25
I could equi e ans-bilaye
mo emen s ha could be ela edly slow.
26
Based on he
concen a ion o he su ac an in he solu ion and he ma ginal
inco po a ion o memb ane p o eins, ou solubiliza ion esul s
showed a low memb ane p o ein occupancy o su ac an
micelles. I implies he p esence o a la ge p opo ion o emp y
micelles and monome s i hese soluble p o eomes a e s udied
by TPP analysis. I is well-s udied ha su ac an s bind o
p o eins in solu ion mainly by elec os a ic, hyd ophobic, and
h-bonding.
18,27
I could be expec ed ha se e al popula ions o
su ac an s, om monome s and micelles o sel -assembly
agg ega es, will be a ailable o possible in e ac ion wi h
p o eins.
18
TTP Analysis and E ec o he Tempe a u e on he
Al e a ion o P o ein Solubili y in Aqueous Micella
Solu ions. Analyzing TPP p o eomes solubilized by he
p e iously desc ibed condi ions, he occu ence o in e ac ions
o globula o memb ane p o ein wi h a di e en subpopula-
ion o su ac an molecules could no be excluded based on
p e ious esul s. The p inciple o he TPP me hod o he
iden i ica ion o a ge s elays on he de ec ion o he al e a ion
o p o ein s abili y unde he mal s imulus caused by p o ein−
chemical compound in e ac ions.
28
The e o e, we aimed o
e alua e i he in e ac ion be ween su ac an s and p o eins
could also be de ec ed in he TPP me hodology as an
al e a ion o p o ein s abili y in solu ion.
29
Fi s , we pe o med
he TPP expe imen and e alua ed he al e a ion in p o ein
solubili y along he he mal shi ange o empe a u es in he
di e en solubiliza ion condi ions. A o al o 3,340 p o eins
we e iden i ied in he samples and hen il e ed acco ding o
he equi emen s o TPP analysis.
28
In he con ol sample in
PBS, he numbe o p o eins iden i ied a he lowes
empe a u e (37 °C) was 1,436 and 1,738 and 1,608 in Igepal
and NP-40S, espec i ely. Conside ing ha he su ac an −
p o ein in e ac ions will espond o di e en p inciples o
globula han memb ane p o eins, he analysis o al e a ion in
solubili y has been p esen ed sepa a ely (Figu e 2).
Fo globula p o eins in he Igepal solu ion, p o eins a e
des abilized a a lowe empe a u e han con ol. An e a ic
pa e n o p o eins changing in solubili y om 53 o 67 °C was
isible on he hea map. In he samples in NP-40S, a su ac an
wi h a CPT a 53 °C, a minimal impac is de ec ed a lowe
empe a u es bu a sha p dec ease a CPT, 53 °C. Fo he
memb ane p o eins in Igepal, he dec ease in p o ein solubili y
is highe han in con ol along he he mal shi ange. Fo NP-
40S, simila o he globula p o eins, he impac o he phase
sepa a ion a ound he CPT empe a u e ma ked a dec ease in
solubili y ha is highe han a any o he empe a u e o he
s udied ange. In summa y, he al e a ion in p o ein solubili y
o p o eins in he Igepal solu ion ollowed e a ic changes. In
he case o NP-40S, he impac o he CPT was deno ed in he
sha p e ec o p o ein des abiliza ion a ha empe a u e
(Figu e 2).
The esul s showed ha he p esence o he nonionic
su ac an in he ex ac ion solu ion has an o e a ching e ec
on he p o eome, al e ing he p o ein solubili y o bo h
globula and memb ane p o eins. This e ec is mo e
p ominen in he p oximi y o he su ac an CPT, ha is,
he empe a u e whe e a phase sepa a ion be ween high and
low su ac an concen a ion is expec ed o be o med.
30
TPP Analysis De e mines he Shi o he P o ein
Mel ing Poin (Tm). The al e a ion o he p o ein solubili y
obse ed in he p e ious expe imen could sugges a ia ion in
he p o ein Tm. The al e a ion o he Tm is he ac o used in
TPP o iden i y p o ein a ge s. The e o e, we pe o med TPP
analysis o de ec di e ences in mel ing poin , plo ed in Figu e
3. All compa isons s a ed wi h 3,340 p o eins, and mel ing
cu es we e la e na owed down based on ou quali y c i e ia
as desc ibed in he me hod.
5
Fo his TPP analysis, he con ol
was equi alen o he sample wi h he ehicle, and he solu ion
wi h he su ac an was de ined as he in e ac o , ha is, he
ea men , acco ding o he TPP R package.
5
The con ol
e sus Igepal analysis showed 52 p o eins, including 23
memb ane p o eins, ha had a di e ence in Tm (Figu e
3A). In con ol e sus NP-40S, 68 p o eins, including 25
memb ane p o eins, ha e al e ed hei Tm (Figu e 3B). The
memb ane p o eins ep esen 44 and 37% o he p o eins wi h
shi ed Tm when he aqueous solu ion con ains su ac an
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micelles. (A lis o all p o eins, mel ing poin s, and p- alue can
be ound in he supplemen a y Table SI−II). When Igepal was
analyzed as he in e ac o , he globula p o eins shi ed hei
Tm o lowe empe a u es han he con ol. The memb ane
p o eins had a wide dis ibu ion o ei he inc eased o
dec eased Tm. In he case o NP-40S, bo h globula and
memb ane p o eins showed lowe Tm han he con ol.
Changes in composi ion and u bidi y o he p o ein
mic oen i onmen a e expec ed wi h he inc ease in empe -
a u e and in he p oximi y o CPT. The p ima y e ec o
inc easing he empe a u e is o educe he deg ee o s uc u e
o wa e nea he micelle su ace, acili a ing he inc eases o
Van de Waals a ac ion due o he closes con ac .
31
This
a ac i e in e ac ion be ween sphe ical micelles o ce ain sizes
acili a es close con ac be ween micelles and leads o s ong
spa ial changes in he mic oen i onmen .
31
Howe e , i is no
easy o model o p edic he he mally dependen a ia ion on
p o ein s abili y in his ype o combined p o eome ha
con ains globula and memb ane p o eins. These esul s
showed a di e en pa e n in he he mally dependen
al e a ion o p o ein solubili y o globula and memb ane
p o eins. Bo h p o ein classes in he p esence o su ac an s a e
desc ibed o ollow di e en p inciples o hei s abiliza ion in
solu ion, and he e o e, mul iple ac o s could lead o p o ein−
su ac an in e ac ions.
32
In he case o globula p o eins, i should be conside ed i
he eason o al e a ion in p o ein solubili y was he
in e ac ion wi h a monome , a micelle, o o he su ac an
sel -assemblies. In some cases, such as human g ow h ac o ,
indi idual monome s could bind o hyd ophobic pa ches ha
could lead o agg ega ion
33
and des abiliza ion, bu o
in e e on-γ, he p o ein is s abilized wi h he in e ac ion
wi h a su ac an .
34
The coope a ion o nonionic monome s
wi h micelles has also been shown o p omo e binding o
globula p o eins and dena u a ion.
35
Se e al me hodologies
could be applied o pu i ied p o eins o e alua e he in e ac ion
o p o eins and small molecules, such as limi ed p o eolysis,
among o he s.
29
Howe e , i would no apply o a p o eome-
wide app oach such as TPP me hodologies. These esul s open
he ques ion o how he TPP analysis could dis inguish
be ween al e a ions in a ge solubili y based on he in e ac ion
wi h a su ac an om al e a ion in solubili y due o
in e ac ions wi h a chemical o a d ug. The e o e, he
applicabili y o he TPP p inciple o he mally induced
al e a ion o p o ein solubili y o a p o eome ex ac ed wi h
nonionic su ac an s could also comp omise he obus
iden i ica ion o globula p o eins as a ge s.
In he case o he memb ane p o eins embedded in micelles,
se e al ac o s could cause he mally induced al e a ion in hei
solubili y, s a ing om he solubiliza ion p ocess ha included
he ans-bilaye mo emen , a sequen ial p ocess, and
complexi y.
26
This p ocess no only equi ed micelliza ion
and he sa u a ion o he memb ane bilaye wi h a su ac an
bu also he ansi ion o he whole bilaye o h ead-like mixed
micelles.
36
The he mally induced changes in he su ac an
o ms desc ibed could also comp omise memb ane p o ein
solubiliza ion. Second, a memb ane p o ein could be
embedded in micelles o di e en sizes. The size o he
micelle is a key ac o in he p ecipi a ion o occupied micelles
by sedimen a ion. The ac ion o lipids om he bilaye
ans e ed in o he micelles would a y om p o ein o
p o ein, and he same p o ein could be embedded in micelles
o di e en sizes.
26
The he mal shi would also al e he
luidi y o lipids inside he micelle, p omo ing he micelle size
ansi ion.
11
These micella s uc u al changes could de e mine
he memb ane p o ein p ecipi a ion wi hou e en any di ec
al e a ion o memb ane p o ein s abili y. Thi d, a la ge
p opo ion o emp y micelles compa ed o he po ion o
micelles embedding in eg al memb ane p o eins should be
expec ed based on he esul s. The e we e e y limi ed
inc eases in memb ane p o ein solubiliza ion wi h he nonionic
su ac an ex ac ion condi ions no mally used in TPP.
13
Consequen ly, emp y micelles and occupied micelles should
coexis in he solu ion and be a ailable o in e ac ions.
Summa izing, i would be e y di icul o de e mine ha any
obse ed al e a ion in he solubili y o a memb ane p o ein
embedded in micelles is exclusi ely caused by a p o ein−
chemical in e ac ion. I should be conside ed ha in he case o
chemical−p o ein in e ac ion wi h memb ane p o eins, i
would ake place on a mino hyd ophilic domain. This ype
o domain equen ly o e s some deg ee o diso de and
lexibili y. Howe e , he la ge pa o he memb ane p o ein,
he hyd ophobic domain, is expec ed o s ay p o ec ed inside a
la ge su ac an micelle ha has shown o be e y sensi i e o
he mally induced al e a ion ha comp omises memb ane
p o ein solubili y.
Analysis o Aqueous Micella Solu ion o Nonionic
Su ac an Used o Solubilize Memb ane P o eins o
TPP Analysis. A e e alua ing p o eomes ex ac ed by
nonionic su ac an solu ions and de e mining a he mally
dependen al e a ion o p o ein solubili y, we aimed o analyze
he e ec o he empe a u e in he su ac an solu ions. The
Igepal and NP-40S solu ions we e p epa ed a he concen-
a ion and p ocedu e u ilized o TPP analysis ha we ha e
p e iously desc ibed. The DLS measu emen om hese
su ac an solu ions was pe o med a en di e en empe -
a u es be ween 37 and 67 °C, as used o TPP. The images
om he DLS wells and DLS measu emen s showed he mally
dependen changes in he size and agg ega ion o he su ac an
in he solu ion.
The DLS well images om he NP-40S solu ion a 49 °C
s a ed o show isual indica ions o s uc u al changes and
u bidi y, bu a lowe empe a u es, a ia ion canno be
Figu e 3. P o eins wi h shi ed mel ing poin s (Tm). P o eins wi h
a ia ions in Tm we e plo ed wi h a mean mel ing poin . (A) Con ol
se as a ehicle o he TPP analysis and Igepal se as an in e ac o .
(B) Con ol se as a ehicle and NP-40S se as an in e ac o . In all
g aphs, he middle ba is he mean alue o wo eplica es and he
e o ba s a e he s anda d de ia ion (SD). MP = memb ane p o eins.
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4038
isually de ec ed. The ins umen could no ake images a
empe a u es highe han 49 °C o sa e y easons (Figu e 4A).
In addi ion, a di e en expe imen simula ing he hea ing and
subsequen ly cooling s ep om he TPP p o ocol was
pe o med wi h bo h he su ac an ex ac ed p o eomes
(Figu e 4B) and wi h su ac an solu ions alone (Figu e
4C,D). The p o eomic samples we e hea ed a 55 and 62 °C
and cooled o RT. The su ac an solu ions we e i s hea ed
un il 55 and 67 °C and cooled a 21 °C. Images om he ubes
con aining he soluble p o eomes e ealed changes in he
u bidi y a e hea ing and cooling (Figu e 4B). Simila ly, he
images om DLS wells om he su ac an solu ions s ill
showed u bidi y a e he cooling (Figu e 4C,D).
The mally induced s uc u al al e a ions o he su ac an
solu ions we e de ec ed om he DLS measu emen s. In he
case o Igepal, DLS measu emen om 37 o 53 °C could be
co ela ed wi h he expec ed micella size and i s g adual
inc ease up o he CPT. Da a poin s we e di icul o egis e
a ound 53−55 °C when he solu ion was likely en e ing in o
clouding. The epo ed CPT o Igepal by he supplie s was
53−67 °C. The DLS measu emen o NP-40S also sugges ed
a he mally dependen inc ease in he micella size ha was
expec ed o be sligh ly la ge han Igepal. The missing da a
poin be ween 48 and 55 °C o NP-40S due o ins umen al
e o connec ed o u bidi y was also close o he expec ed
CPT a ound 45−50 °C. Bo h su ac an solu ions showed a
sha p inc ease in he DLS measu ed size om 55 o 65 °C,
which we e se e al o de s o magni ude abo e a micella size
bu could co espond o complex su ac an sel -assembly
beha io
37
(Figu e 5A,B). The DLS measu emen om he
hea ing and cooling expe imen s wi h su ac an solu ions
co obo a ed he p esence o la ge su ac an sel -assemblies
ha did no e e a e 3 min a 21 °C (Figu e 5A,B blue
ci cles).
The expec ed changes in he s uc u e o he nonionic
su ac an in an aqueous solu ion a e de ined by he
concen a ion, solu ion, and empe a u e.
19
Ou s udy
e alua ed he di ec e ec o he empe a u e by main aining
he es o he pa ame e s cons an . The su ac an solu ions a
concen a ion and he inc eases in empe a u e used o TPP
analysis showed p ominen s uc u al changes. These changes
could be ela ed o micella g ow h, clouding, and he
accumula ion o la ge s uc u es o su ac an sel -assemblies
and did no e e a e he cooling s ep.
The solubili y o nonionic su ac an s in wa e has been
desc ibed as a delica e balance be ween hyd ophobic and
Figu e 4. Images om he mal al e a ion in su ac an solu ions and
p o eins ex ac ed wi h su ac an solu ions. (A) Images om he
bo om o he DLS wells hea ed a 49 °C. Samples le - o- igh ,
con ol PBS, 0.4% Igepal in PBS, and 0.4% NP-40S in PBS. (B)
Images om ubes con aining he soluble p o eome ex ac ed wi h
Igepal (le ) and NP-40S ( igh ) a e di e en he mal ea men s:
hea ing o 55 °C, hea ing o 62 °C, hea ing o 55 °C, and cooling o
RT, and hea ing o 62 °C ollowed by cooling o RT. (C) Images
om DLS wells con ol PBS e sus he wo su ac an solu ions
hea ed o 55 °C and hen cooled o 21 °C. (D) Images om DLS
wells con ol PBS e sus he wo su ac an solu ions hea ed o 67 °C
and hen cooled o 21 °C. The expe imen s we e pe o med wi h and
wi hou silicone laye ing o e alua e any possible e ec s o
e apo a ion. The samples wi h longi udinal sec ions a e composi es
whe e he le side co esponded o he expe imen wi hou silicone
laye ing and he igh wi h co e ing.
Figu e 5. DLS measu emen o he nonionic su ac an solu ions
exposed o 10 s epwise inc eases o empe a u e. (A) Igepal solu ions
exposed o 37−67 °C. (B) NP-40S solu ions exposed o 37−67 °C.
Whi e ci cles ep esen DLS measu emen , c ossed ci cles ep esen
no-measu emen due o au o-a enua ing e o a he DLS ins umen ,
and blue ci cles we e DLS measu emen s ob ained om samples ha
we e sepa a ely hea ed a 55 o 67 °C (abo e CPTs) and cooled o 21
°C. Se e al da a poin s could no be ob ained due o echnical
easons.
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hyd ophilic in e ac ions ha assis in main aining he
su ac an molecules’ solubili y. The e ec s o he inc ease in
empe a u e can easily modi y hese in e ac ions and dec ease
he su ac an solubili y. Small changes in he e ec i e
in e ac ion be ween he su ac an and wa e cause d as ic
e ec s such as phase o ma ion.
37
A clouding, he sepa a ion
be ween he su ac an - ich and su ac an -lean s a s wi h
changes in u bidi y ha can be mac oscopically obse ed.
19,37
This is an endo he mic ansi ion a ibu ed o he con o ma-
ional ea angemen o he assembled su ac an molecules,
especially he pola head g oups, and i s changes in he
in e ac ion wi h wa e molecules.
38
Acco ding o he models
p oposed, he inc ease o empe a u e in he ansi ion phase
could lead o a emo al o wa e molecules ha we e associa ed
wi h he pola head g oups a he bonds, b eaking he
in e molecula hyd ogen bonds. Losing he sol a ed molecules
may induce con o ma ional changes in he micelles wi h a
dec ease in olume, causing a dec ease in s abili y a he
sphe ical shape. The e o e, he micelles could end o adop a
mo e plana a angemen ha would s abilize he su ac an
molecules in he new en i onmen .
19
The educ ion o he
micella cu a u e could also acili a e he associa ion o
micelles o micelles wi h he o ma ion o long cylind ical
micelles wi h bigge olumes. These la ge and elonga ed
micelles would equi e lowe cen i uga ion o ce o sedimen
han he smalle micelles ha we e mainly p esen a lowe
empe a u es. The e o e, he s uc u al changes o su ac an
solu ions wi h he empe a u e a e expec ed o al e he
p ecipi a ion pa e n o globula p o eins in e ac ing wi h a
su ac an and memb ane p o ein in su ac an micelles. I
should be eminded ha in TPP me hods, he cen i uga ion
o ce is applied o disc imina e be ween soluble and insoluble
p o eins and o iden i y he chemical− a ge in e ac ion.
Mic oscopic Imaging o P o eins Exposed o TPP
The mal T ea men . Mic oscopic imaging o he p o eomes
exposed o hea was p epa ed ollowing he TPP me hod o
isualize mic oscopical changes in he aqueous micella
solu ion in he p esence o p o eins. Samples we e hea ed o
67 °C o 3 min and imaged using ligh mic oscopy wi hin he
i s 3 min, ollowing he hea ing pe iod equi ed in TPP
me hodology. In he images, a ying sizes o p o ein agg ega es
can be obse ed, as illus a ed wi h he black a ows.
The con ol con ained small p o ein agg ega es loca ed close
o each o he , he Igepal sample con ained sligh ly la ge
p o ein agg ega es, and NP-40S con ained he la ges p o ein
agg ega es, which a e loca ed u he apa (Figu e 6). These
mic oscopic images showing la ge su ac an and p o ein
agg ega es con i m ha he p o eomic samples exposed o he
TPP highes empe a u e ollowed a simila pa e n compa ed
o he su ac an solu ions.
■CONCLUSIONS
The use o a nonionic su ac an in he p o eome analyzed by
TPP in oduces pa ame e s ha could al e he p o ein
solubili y independen ly om he chemical− a ge in e ac ion.
We showed ha he s anda d p o ocol u ilized did no acili a e
he solubiliza ion o in eg al memb ane p o eins. Howe e ,
soluble p o eomes in su ac an solu ions could con ain a la ge
p opo ion o emp y micelles a ailable o in e ac ion wi h
bo h globula and memb ane p o eins du ing he TPP
wo k low. The e ec s o hese in e ac ions we e indi ec ly
de e mined by he b oad al e a ion in p o ein solubili y in he
s udied p o eomes, shi o he p o ein Tm, and mic oscopic
imaging o p o eins o ming agg ega es. The applica ion o he
TPP he mal ea men o he su ac an solu ion demon-
s a ed ha i ollowed he expec ed s uc u al changes,
including he inc ease in he micella size and o ma ion o
o he sel -assembly s uc u es a he mac oscopic size. Ou
esul s lead o he explo a ion o he mally independen
solubiliza ion s a egies o assu e a con iden memb ane
p o ein a ge iden i ica ion by a high- h oughpu me hod
based on p o eome solubili y al e a ion.
■ASSOCIATED CONTENT
*
sı Suppo ing In o ma ion
The Suppo ing In o ma ion is a ailable ee o cha ge a
h ps://pubs.acs.o g/doi/10.1021/acs.analchem.2c04500.
De ailed expe imen al p o ocols, including issue p o ein
ex ac ion, p o eomic sample p epa a ion, nano liquid
ch oma og aphy-mass spec ome y (LC-MS/MS)
me hods, TPP da a analysis, and a lis o all p o eins,
mel ing poin s, and p- alues (PDF)
■AUTHOR INFORMATION
Co esponding Au ho
Susana C is obal −Depa men o Biomedical and Clinical
Sciences, Cell Biology, Facul y o Medicine, Linköping
Uni e si y, Linköping 581 85, Sweden; Ike basque, Basque
Founda ion o Sciences, Depa men o Physiology, Facul y o
Medicine, and Nu sing, Uni e si y o he Basque Coun y
UPV/EHU, Leioa 489 40, Spain; o cid.o g/0000-0002-
3894-2218; Phone: +46-730385867;
Email: [email p o ec ed]
Figu e 6. Mic oscopy isualiza ion o p o eins ex ac ed wi h PBS (con ol), Igepal, o NP-40S solu ions. The p o eomic smaples we e analyzed
a e ollowing a he mal s ep om he TPP me hodology, hea ed o 67 °C and isualized wi hin he i s 3 min. The la ges image is me ged iles
con aining a scale ba o 100 μm. The smalle image is one ile wi h a scale ba o 30 μm. The black a ow shows one example o a p o ein
agg ega e.
Analy ical Chemis y pubs.acs.o g/ac A icle
h ps://doi.o g/10.1021/acs.analchem.2c04500
Anal. Chem. 2023, 95, 4033−4042
4040
Au ho s
Emmanuel Be lin −Depa men o Biomedical and Clinical
Sciences, Cell Biology, Facul y o Medicine, Linköping
Uni e si y, Linköping 581 85, Sweden; P esen
Add ess: E.B.: Depa men o Chemis y and Molecula
Biology, Uni e si y o Go henbu g, Go henbu g 41 296,
Sweden
Ve onica Lizano-Fallas −Depa men o Biomedical and
Clinical Sciences, Cell Biology, Facul y o Medicine, Linköping
Uni e si y, Linköping 581 85, Sweden
Ana Ca asco del Amo −Depa men o Biomedical and
Clinical Sciences, Cell Biology, Facul y o Medicine, Linköping
Uni e si y, Linköping 581 85, Sweden
Ola z F esnedo −Depa men o Physiology, Facul y o
Medicine, and Nu sing, Uni e si y o he Basque Coun y
UPV/EHU, Leioa 489 40, Spain
Comple e con ac in o ma ion is a ailable a :
h ps://pubs.acs.o g/10.1021/acs.analchem.2c04500
Au ho Con ibu ions
⊥
E.B. and V.L.-F. ha e equally con ibu ed and sha ed he i s
au ho posi ion. E.B. has pe o med mos o he labo a o y
wo k and analyzed he da a, and V.L.-F has di ec ly supe ised
he expe imen s and con ibu ed o he da a analyzed. A.C.A.
conduc ed some addi ional expe imen s. O.F. has p o ided
biological samples and con ibu ed o he discussion o he
esul s and he expe imen al design o he expe imen s. S.C.
gene a ed he idea and designed he s udy, supe ised he
analysis o he expe imen al wo k, discussed and in e p e ed
he esul s, w o e, edi ed he manusc ip , and was esponsible
o unding acquisi ion. All he au ho s ha e con ibu ed o
discussions and modi ica ions o he manusc ip and app o ed
i .
Funding
This wo k has been pe o med wi h unding om he ERA-
NET Ma ine Bio echnology p ojec CYANOBESITY, which is
co ounded by FORMAS, Sweden, g an no. 2016-02004
(S.C.); he p ojec GOLIATH ha has ecei ed unding
om he Eu opean Union’s Ho izon 2020 esea ch and
inno a ion p og am unde g an ag eemen No 825489
(S.C.); IKERBASQUE, Basque Founda ion o Science
(S.C.); Basque Go e nmen Resea ch G an IT-971-16 and
IT-476-22 (S.C.); Magnus Be g alls Founda ions (S.C.),
VINNOVA No 2021-04909 (S.C.), and he g an o doc o al
s udies OAICE-75-2017 Wo ld Bank coun e pa - Uni e si y
o Cos a Rica (V.L.-F.).
No es
The au ho s decla e no compe ing inancial in e es .
■ACKNOWLEDGMENTS
All o he mass spec ome y analysis has been pe o med wi h
ins umen a ion a he LiU MS acili y.
■ABBREVIATIONS
MS mass spec ome y
TPP he mal p o eome p o iling
NP-40S nonyl-phenyl-poly(e hylene glycol)
Igepal Igepal CA-630
NP-40 oc yl-phenoxy(polyoxye hylene)e hanol
CPT cloud poin empe a u e
DLS dynamic ligh sca e ing
Tm mel ing poin
TSA he mal shi assay
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