In e na ional Jou nal o
Molecula Sciences
A icle
P o eomic Analysis in Mo quio A Cells T ea ed wi h
Immobilized Enzyma ic Replacemen The apy on
Nanos uc u ed Lipid Sys ems
J. Víc o Ál a ez 1,2,3,†, Susana B. B a o 4,†, Ma ía Ga cía-Vence 4, Ma ía J. De Cas o 2,
As e ia Luza do 5,6 , C is óbal Colón2, Shunji Toma su 3, F ancisco J. O e o-Espina 1,6,* and
Ma ía L. Couce 2,*
1
Depa men o Pha macology, Pha macy and Pha maceu ical Technology, School o Pha macy, Campus Vida,
Uni e si y o San iago de Compos ela, 15872 San iago de Compos ela, Spain;
jose ic o [email p o ec ed]g
2Depa men o Fo ensic Sciences, Pa hology, Gynecology and Obs e ics, Pedia ics, Neona ology Se ice,
Depa men o Paedia ics, Hospi al Clínico Uni e si a io de San iago de Compos ela, Heal h Resea ch
Ins i u e o San iago de Compos ela (IDIS), CIBERER, Me abERN, 15706 San iago de Compos ela, Spain;
[email p o ec ed] (M.J.D.C.); c is obal.colon.meje as@se gas.es (C.C.)
3Skele al Dysplasia Lab Nemou s Biomedical Resea ch Nemou s/Al ed I. duPon Hospi al o Child en,
1600 Rockland Road, Wilming on, DE 19803, USA; [email p o ec ed]g
4P o eomic Pla o m, Heal h Resea ch Ins i u e o San iago de Compos ela (IDIS), Hospi al Clínico
Uni e si a io de San iago de Compos ela, 15706 San iago de Compos ea, Spain; [email p o ec ed] (S.B.B.);
[email p o ec ed] (M.G.-V.)
5Depa men o Pha macology, Pha macy and Pha maceu ical Technology, School o Sciences,
Campus de Lugo, Uni e si y o San iago de Compos ela, 27002 Lugo, Spain; [email p o ec ed]
6Pa aquasil Pla o m, Heal h Resea ch Ins i u e o San iago de Compos ela (IDIS), Hospi al Clínico
Uni e si a io de San iago de Compos ela, 15706 San iago de Compos ela, Spain
*Co espondence: [email p o ec ed] (F.J.O.-E.); [email p o ec ed] o
[email p o ec ed] (M.L.C.); Tel.: +34-981563100 (ex . 24142; ex . 14878) (F.J.O.-E.);
+34-981951100 (M.L.C.)
†These au ho s con ibu ed equally o his wo k.
Recei ed: 22 Augus 2019; Accep ed: 13 Sep embe 2019; Published: 18 Sep embe 2019
Abs ac :
Mo quio A synd ome, o mucopolysaccha idosis ype IVA (MPS IVA), is a lysosomal
s o age disease due o mu a ions in he N-ace ylgalac osamine-6-sul a ase (GALNS) gene. Sys emic
skele al dysplasia and he ela ed clinical ea u es o MPS IVA a e due o dis up ion o ca ilage and
i s ex acellula ma ix, leading o an imbalance o g ow h. Enzyme eplacemen he apy (ERT) wi h
ecombinan human GALNS, alpha elosul ase, p o ides a sys emic ea men . Howe e , his he apy
has a limi ed impac on skele al dysplasia because he in used enzyme canno pene a e ca ilage and
bone. The e o e, an al e na i e he apeu ic app oach o each he ca ilage is an unme challenge.
We ha e de eloped a new d ug deli e y sys em based on a nanos uc u e lipid ca ie wi h he
capaci y o immobilize enzymes used o ERT and o a ge he lysosomes. This s udy aimed o
assess he e ec o he encapsula ed enzyme in his new deli e y sys em, using
in i o
p o eomic
echnology. We ound a g ea e in e naliza ion o he enzyme ca ied by nanopa icles inside he cells
and an imp o emen o cellula p o ein ou es p e iously impai ed by he disease, compa ed wi h
con en ional ERT. This is he i s quali a i e and quan i a i e p o eomic assay ha demons a es he
ad an ages o a new deli e y sys em o imp o e he MPS IVA ERT.
Keywo ds: enzyme eplacemen he apy; lysosomal diso de s; nanopa icles; p o eomics
In . J. Mol. Sci. 2019,20, 4610; doi:10.3390/ijms20184610 www.mdpi.com/jou nal/ijms
In . J. Mol. Sci. 2019,20, 4610 2 o 22
1. In oduc ion
Mo quio A synd ome, o mucopolysaccha idosis ype IVA (MPS IVA, OMIM #253000), is an
au osomal ecessi e disease, caused by mu a ions in he N-ace ylgalac osamine-6-sul a ase (GALNS),
gene which esul in de icien ac i i y o N-ace ylgalac osamine-6-sul a ase (GALNS, E.C.3.1.6.4),
an enzyme ha deg ades glycosaminoglycan (GAG) ke a an sula e (KS) and chond oi in-6-sul a e
(C6S) [
1
,
2
]. The widesp ead accumula ion o GAG, p ima ily in chond ocy es, and i s ex acellula
ma ix, leads o p og essi e cellula damage and o gan dys unc ion in he bone and ca ilage.
The classical pheno ype is cha ac e ized by sys emic skele al dysplasia, including a sho s a u e
and neck, ce ical ins abili y and spinal co d comp ession, acheal obs uc ion, p ominen ches ,
kyphoscoliosis, laxi y o join s, hip dysplasia, knock knee, e c. [
3
]. In se e e o ms, espi a o y ailu e
is he p ima y cause o dea h du ing he second and hi d decade o li e i un ea ed [4,5].
The e a e wo ea men s o MPS IVA pa ien s in clinical p ac ice: enzyme eplacemen he apy
(ERT) and hema opoie ic s em cell ansplan a ion. ERT wi h elosul ase alpha, a ecombinan human
GALNS, is an es ablished s a egy o ea ing MPS IVA [
1
,
6
,
7
]. Howe e , clinical ials ha e shown
sca ce imp o emen in g ow h and skele al dysplasia [
8
–
11
]. In pa ien s wi h MPS IVA, es ic i e
and obs uc i e lungs a e due o an ana omical imbalance in g ow h ( achea and essels g ow while
he spine and ho acic bones s op g owing), leading o his li e- h ea ening issue. ERT canno a ec
bone de o mi y in he spine, ibs and s e num, and c owded ho acic ca i y. The e o e, ERT may
p o ide a limi ed impac on pulmona y unc ion due o he emaining issue o skele al dysplasia
( es ic i e lung). A ecen long- e m s udy o MPS IVA wi h ERT has shown ha he e was a global
educ ion in s a ic spi ome y alues in all subjec s wi h ERT, as well as ca dio espi a o y unc ion
as assessed by 6MWT [
12
,
13
]. Simila o o he ERTs o lysosomal s o age diseases, elosul ase alpha
has se e al pi alls: (i) apid clea ance om ci cula ion and a sho hal -li e (2 min in mice; 35 min
in humans) [
10
,
12
,
14
], (ii) limi ed pene a ion in he a ascula ca ilage, and (iii) immunological
esponse agains he in used enzyme [12,15,16]. Thus, he apies o esol ing bone lesions emain an
unme need, and an al e na i e he apeu ic app oach is equi ed o o e come such limi a ions. In his
ega d, one possible solu ion is a nanopa icula sys em ha can p o ec he enzymes agains hei
deg ada ion o inac i a ion, minimize he associa ed immunological eac ions, and inc ease cellula
in e naliza ion o achie e maximum e icacy o he ea men [
17
]. Biodeg adable nanopa icles o a
di e en na u e, such as polylac ic-co-glycolic acid, ha e been used in ERT. Some s udies ha e also
shown he use ulness o solid lipid nanopa icles [
8
,
12
,
15
,
16
] o achie e he in e naliza ion o molecules,
such as ca alase, leup olide, o insulin [
18
–
21
], o he cellula in e io h ough hei di ec endocy osis
owa ds lysosomes. One o he main limi a ions o hese sys ems is hei low capaci y o immobilize
and anspo a p o ein in o he lipid co e. Some expe imen s ha e been ca ied ou o abso b he
p o ein on he su ace o he lipid colloidal ca ie s, bu in he case o enzymes, his app oach can ha e
d awbacks ela ed o ins abili y and he p oduc ion o an ibodies. Consequen ly, o mula ion design is
a c i ical equi emen o s abilize a high payload o he enzyme. We ha e ecen ly de eloped a new
d ug deli e y sys em o immobilize enzymes used o ERT and o a ge he lysosomes [nanos uc u e
lipid ca ie (NLC), pa en egis e ed numbe PCT/EP2019/068629].
P o eomics allows no only he iden i ica ion bu also he quan i ica ion o he o e all p o eins
p esen in a cell, issue, o o ganism in con inuous change. One o he i s p o eomic app oaches
includes a one o wo-dimensional polyac ylamide gel sys em using sodium dodecyl sul a e (1D
SDS-PAGE and 2D SDS-PAGE) and p o ides a powe ul ool o moni o p o ein pu i ica ion and apidly
p o ile he global p o ein exp ession and pos ansc ip ional modi ica ions [
22
]. Al hough 1D and
2D-PAGE a e common p o eomic app oaches o high h oughpu sc eening o pu a i e bioma ke s
in se e al diso de s, pa icula ly in animal s udies [
23
,
24
], hey ha e no been used in lysosomal
diseases. On he o he hand, mass spec ome y (MS) is a bene icial p o eomic echnology ha allows
p o eomic pa e n isualiza ion and he iden i ica ion o p o eins [
25
,
26
] and hei pos - ansc ip ional
modi ica ions, such as phospho yla ion, glycosyla ion, simila o 2D-Page app oach [
27
]. In he las
decade,MShasmadesigni ican p og essbyinc easing henumbe o iden i iedp o eins[
26
]. The e o e,
In . J. Mol. Sci. 2019,20, 4610 3 o 22
liquid ch oma og aphy (LC) coupled wi h MS is widely used o sea ching disease bioma ke s [
26
] in
samples such as biological luids, issues, cells, e c. [28,29].
Mos quan i a i e me hods in ol e labeling p o eins wi h hea y and ligh s able-iso ope pai s
(SILAC, I-TRAQ). O he quan i a ion echniques elimina e labeling (label- ee) and ely on ad anced
so wa e analysis as a new echnology named SWATH-MS (sequen ial window acquisi ion o all
heo e ical mass spec a) [
30
,
31
]. These me hods measu e he ela i e concen a ions o pep ide analy es
wi hin wo o mo e samples. In con as , absolu e quan i a ion echniques use in e nal s anda d
pep ides ha ha e been syn he ically p epa ed o selec ed o mul iple eac ion moni o ing (selec ed
eac ion moni o ing o mul iple eac ion moni o ing, espec i ely) analysis [25].
In gene al, disease s a es in ol e al e a ions in p o ein exp ession le els. The e o e, p o eomics
p o ides impo an biological in o ma ion ha con ibu es o he ad ance in ou unde s anding
o pa hophysiological mechanisms and aid in he iden i ica ion o no el bioma ke s o di e en
diseases [
30
,
32
,
33
]. Speci ic bioma ke s iden i ied om p o eomics may be use ul in diagnosis,
p ognosis, and as e alua o s o ea men ou comes [34–42].
In his s udy, we ha e analyzed he e ec s o p o ein exp ession le els in he MPS IVA ib oblas s
by p o iding he immobilized and ee enzyme (GALNS) and by using quali a i e and quan i a i e
p o eomic echniques.
2. Resul s
2.1. Quali a i e Analysis Liquid Ch oma og aphy—Mass Spec ome y (DDA-LC-MS/MS)
By using DDA-LC-MS/MS, we iden i ied 287 o 1623 p o eins in un ea ed cells and 586 o
1817 p o eins in ib oblas s ea ed wi h NLC +ERT o ERT alone (Table 1).
Table 1. The numbe o p o eins iden i ied in he quali a i e (DDA-LC-MSMS) assay.
Numbe o P o eins.
1s Analysis
Numbe o P o eins.
2nd Analysis
Numbe o P o eins.
3 d Analysis
Samples No
T ea
NLC +
ERT ERT No
T ea
NLC +
ERT ERT No
T ea
NLC +
ERT ERT
HC 683 1395 1612 1561 1668 919 737 1183
S.1 MoC 730 960 923 1304 1623 1817
S.2 MoC 1220 1255 832 1022 793 1078
S.3 MoC 793 1658 1251 1594 1419 1104
S.4 MoC 611 1445 287 870 1403 586
No e: All he assays we e made in iplica e. HC: heal hy cells; ea : ea men ; NLC: nanos uc u e lipid ca ie ;
LC: lipid ch oma og aphy; ERT: Enzyme eplacemen he apy; MoC: Mo quio A cells; S.1: Sample 1.
To ob ain a be e ep esen a ion o all he p o eins iden i ied in each ea men , we selec ed
only hose ha we e p esen in a leas 2 o he eplica es pe ea men (Figu e 1and Supplemen a y
Figu e S1).
To analyze he o igin o he iden i ied p o eins unde no ea men , NLC +ERT, and ERT alone,
we used FunRich analysis. The majo i y o p o eins in all he samples we e ound o be sou ced om
ou di e en cellula componen s: mi ochond ion, lysosomal memb ane, lysosome, and endosome
(Figu e 2A). In heal hy cells (HC) wi h ERT (HC +ERT), we ound an inc ease in a small pe cen age
o p o eins compa ed wi h un ea ed heal hy cells (UHC), pa icula ly o he mi ochond ion and
endosome. In he compa ison be ween un ea ed Mo quio A cells (UMoC) s. Mo quio A cells wi h
ERT (MoC +ERT), no changes (o a minimal change) we e ound (Figu e 2B and Supplemen a y
Figu e S2). When we compa ed HC s. HC wi h NLC +ERT (HC NLC +ERT) (Figu e 2C and
Supplemen a y Figu e S2), we ound only a small dec ease in he pe cen age o mi ochond ion and
endosome. When we compa ed UMoC s. he MoC NLC +ERT (Figu e 2D and Supplemen a y
In . J. Mol. Sci. 2019,20, 4610 4 o 22
Figu e S2), small di e ences in he pe cen age o p o eins (a sligh inc ease in mi ochond ion and
endosome and a sligh dec ease in lysosomal memb ane and lysosome) we e ound.
In . J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 3 o 22
Mos quan i a i e me hods in ol e labeling p o eins wi h hea y and ligh s able-iso ope pai s
(SILAC, I-TRAQ). O he quan i a ion echniques elimina e labeling (label- ee) and ely on ad anced
so wa e analysis as a new echnology named SWATH-MS (sequen ial window acquisi ion o all
heo e ical mass spec a) [30,31]. These me hods measu e he ela i e concen a ions o pep ide
analy es wi hin wo o mo e samples. In con as , absolu e quan i a ion echniques use in e nal
s anda d pep ides ha ha e been syn he ically p epa ed o selec ed o mul iple eac ion moni o ing
(selec ed eac ion moni o ing o mul iple eac ion moni o ing, espec i ely) analysis [25].
In gene al, disease s a es in ol e al e a ions in p o ein exp ession le els. The e o e, p o eomics
p o ides impo an biological in o ma ion ha con ibu es o he ad ance in ou unde s anding o
pa hophysiological mechanisms and aid in he iden i ica ion o no el bioma ke s o di e en
diseases [30,32,33]. Speci ic bioma ke s iden i ied om p o eomics may be use ul in diagnosis,
p ognosis, and as e alua o s o ea men ou comes [34–42].
In his s udy, we ha e analyzed he e ec s o p o ein exp ession le els in he MPS IVA
ib oblas s by p o iding he immobilized and ee enzyme (GALNS) and by using quali a i e and
quan i a i e p o eomic echniques.
2. Resul s
2.1. Quali a i e Analysis Liquid Ch oma og aphy – Mass Spec ome y (DDA-LC-MS/MS)
By using DDA-LC-MS/MS, we iden i ied 287 o 1623 p o eins in un ea ed cells and 586 o 1817
p o eins in ib oblas s ea ed wi h NLC+ERT o ERT alone (Table 1).
Table 1. The numbe o p o eins iden i ied in he quali a i e (DDA-LC-MSMS) assay.
Numbe o p o eins. 1s Analysis Numbe o P o eins.
2nd Analysis
Numbe o P o eins.
3 d Analysis
Samples No
ea NLC+ERT ERT No
ea NLC+ERT ERT No
ea NLC+ERT ERT
HC 683 1395 1612 1561 1668 919 737 1183
S.1 MoC 730 960 923 1304 1623 1817
S.2 MoC 1220 1255 832 1022 793 1078
S.3 MoC 793 1658 1251 1594 1419 1104
S.4 MoC 611 1445 287 870 1403 586
No e: All he assays we e made in iplica e. HC: heal hy cells; ea : ea men ; NLC:
nanos uc u e lipid ca ie ; LC: lipid ch oma og aphy; ERT: Enzyme eplacemen he apy;
MoC: Mo quio A cells; S.1: Sample 1.
To ob ain a be e ep esen a ion o all he p o eins iden i ied in each ea men , we selec ed only
hose ha we e p esen in a leas 2 o he eplica es pe ea men (Figu e 1 and Supplemen a y
Figu e S1).
Figu e 1.
Venn diag am om he iplica es s udies o p o eins in Mo quio A cells (Sample 1) a e 24 h
o NLC +ERT ea men . The common p o eins iden i ied in 2 o 3 o he eplica es a e shown.
In . J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 4 o 22
Figu e 1. Venn diag am om he iplica es s udies o p o eins in Mo quio A cells (Sample
1) a e 24 h o NLC+ERT ea men . The common p o eins iden i ied in 2 o 3 o he
eplica es a e shown.
To analyze he o igin o he iden i ied p o eins unde no ea men , NLC+ERT, and ERT alone,
we used FunRich analysis. The majo i y o p o eins in all he samples we e ound o be sou ced om
ou di e en cellula componen s: mi ochond ion, lysosomal memb ane, lysosome, and endosome
(Figu e 2A). In heal hy cells (HC) wi h ERT (HC+ERT), we ound an inc ease in a small pe cen age
o p o eins compa ed wi h un ea ed heal hy cells (UHC), pa icula ly o he mi ochond ion and
endosome. In he compa ison be ween un ea ed Mo quio A cells (UMoC) s. Mo quio A cells wi h
ERT (MoC+ERT), no changes (o a minimal change) we e ound (Figu e 2B and Supplemen a y
Figu e 2). When we compa ed HC s. HC wi h NLC+ERT (HC NLC+ERT) (Figu e 2C and
Supplemen a y Figu e 2), we ound only a small dec ease in he pe cen age o mi ochond ion and
endosome. When we compa ed UMoC s. he MoC NLC+ERT (Figu e 2D and Supplemen a y Figu e
S2), small di e ences in he pe cen age o p o eins (a sligh inc ease in mi ochond ion and endosome
and a sligh dec ease in lysosomal memb ane and lysosome) we e ound.
Figu e 2. FunRich unc ional analysis esul s o exp essed p o eins om cellula
componen s. (A) HC and HC + ERT. (B) UMoC (Sample 2) and MoC + ERT (Sample 2). (C)
HC and HC wi h NLC+ ERT. (D) UMoC (Sample 1) and MoC wi h NLC+ERT (Sample 1).
Rega ding he biological p ocess o he mi ochond ion, we ound changes in he p o eins o be
ela ed o he in e ac ion be ween he mi ochond ia and he lysosome (Figu e 3). These changes we e
mo e p ominen in he cells ea ed wi h NLC+ERT (Figu e 3A) o ERT (Figu e 3B). The analysis
indica es ha he exp ession le el o p o eins ela ed o cell edox homeos asis, he esponse o
oxida i e s ess, he esponse o calcium ion, ans e in anspo , amino acid anspo , and
mi ochond ion o lysosome anspo we e dec eased in bo h ea men s, while he exp ession le els
o p o eins ela ed o a y acid β-oxida ion we e inc eased.
Figu e 2.
FunRich unc ional analysis esul s o exp essed p o eins om cellula componen s. (
A
) HC
and HC +ERT. (
B
) UMoC (Sample 2) and MoC +ERT (Sample 2). (
C
) HC and HC wi h NLC +ERT.
(D) UMoC (Sample 1) and MoC wi h NLC +ERT (Sample 1).
Rega ding he biological p ocess o he mi ochond ion, we ound changes in he p o eins o be
ela ed o he in e ac ion be ween he mi ochond ia and he lysosome (Figu e 3). These changes we e
mo e p ominen in he cells ea ed wi h NLC +ERT (Figu e 3A) o ERT (Figu e 3B). The analysis
indica es ha he exp ession le el o p o eins ela ed o cell edox homeos asis, he esponse o oxida i e
s ess, he esponse o calcium ion, ans e in anspo , amino acid anspo , and mi ochond ion o
lysosome anspo we e dec eased in bo h ea men s, while he exp ession le els o p o eins ela ed
o a y acid β-oxida ion we e inc eased.
In . J. Mol. Sci. 2019,20, 4610 5 o 22
In . J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 5 o 22
Figu e 3. FunRich unc ional analysis esul s o he exp essed p o eins om he me abolic
p ocess o mi ochond ion. (A) Compa ison be ween un ea ed and ea ed o 24 h wi h
NLC + ERT in HC and MoC. (B) Compa ison be ween un ea ed and ea ed o 24 h wi h
ERT alone in HC and MoC.
When we analyzed he biological p ocess o he endosome (Figu e 4), he changes in he p o eins
in ol ed in he endosome- o-lysosome anspo and i on ion homeos asis we e obse ed. We ound
an inc ease in endosome- o-lysosome anspo in bo h HC and MoC wi h NLC+ERT (Figu e 4A) bu
only in HC wi h ERT alone (Figu e 4B). In he case o he i on ion homeos asis anspo , we ound a
signi ican inc ease in MoC wi h bo h NLC+ERT and ERT alone (Figu e 4A,B) and only a small
inc ease in HC wi h ERT (Figu e 4B).
Figu e 4. FunRich unc ional analysis esul s o exp essed p o eins om he me abolic
p ocess o he endosome. (A) compa ison be ween un ea ed and ea ed o 24 h wi h NLC
+ ERT in HC and MoC; (B) compa ison be ween un ea ed and ea ed o 24 h wi h ERT in
HC and MoC.
In he lysosomal memb ane (Figu e 5), we obse ed a dec ease o he exp ession le el in he
p o eins ela ed o a ge ing he lysosome and o he lysosomal memb ane o ganiza ion in NLC+ERT
(Figu e 5A). Howe e , in ERT ea men s, we only ound a dec ease in he lysosomal memb ane
(Figu e 5B). Mo eo e , i should be no ed ha in HC, bo h HC alone o wi h ea men (NLC+ERT o
ERT), we ound ha he a ge ed lysosome and lysosomal memb ane o ganiza ion p o ein le els
dec ease (Figu e 5A,B).
Figu e 3.
FunRich unc ional analysis esul s o he exp essed p o eins om he me abolic p ocess o
mi ochond ion. (
A
) Compa ison be ween un ea ed and ea ed o 24 h wi h NLC +ERT in HC and
MoC. (B) Compa ison be ween un ea ed and ea ed o 24 h wi h ERT alone in HC and MoC.
When we analyzed he biological p ocess o he endosome (Figu e 4), he changes in he p o eins
in ol ed in he endosome- o-lysosome anspo and i on ion homeos asis we e obse ed. We ound
an inc ease in endosome- o-lysosome anspo in bo h HC and MoC wi h NLC +ERT (Figu e 4A) bu
only in HC wi h ERT alone (Figu e 4B). In he case o he i on ion homeos asis anspo , we ound
a signi ican inc ease in MoC wi h bo h NLC +ERT and ERT alone (Figu e 4A,B) and only a small
inc ease in HC wi h ERT (Figu e 4B).
In . J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 5 o 22
Figu e 3. FunRich unc ional analysis esul s o he exp essed p o eins om he me abolic
p ocess o mi ochond ion. (A) Compa ison be ween un ea ed and ea ed o 24 h wi h
NLC + ERT in HC and MoC. (B) Compa ison be ween un ea ed and ea ed o 24 h wi h
ERT alone in HC and MoC.
When we analyzed he biological p ocess o he endosome (Figu e 4), he changes in he p o eins
in ol ed in he endosome- o-lysosome anspo and i on ion homeos asis we e obse ed. We ound
an inc ease in endosome- o-lysosome anspo in bo h HC and MoC wi h NLC+ERT (Figu e 4A) bu
only in HC wi h ERT alone (Figu e 4B). In he case o he i on ion homeos asis anspo , we ound a
signi ican inc ease in MoC wi h bo h NLC+ERT and ERT alone (Figu e 4A,B) and only a small
inc ease in HC wi h ERT (Figu e 4B).
Figu e 4. FunRich unc ional analysis esul s o exp essed p o eins om he me abolic
p ocess o he endosome. (A) compa ison be ween un ea ed and ea ed o 24 h wi h NLC
+ ERT in HC and MoC; (B) compa ison be ween un ea ed and ea ed o 24 h wi h ERT in
HC and MoC.
In he lysosomal memb ane (Figu e 5), we obse ed a dec ease o he exp ession le el in he
p o eins ela ed o a ge ing he lysosome and o he lysosomal memb ane o ganiza ion in NLC+ERT
(Figu e 5A). Howe e , in ERT ea men s, we only ound a dec ease in he lysosomal memb ane
(Figu e 5B). Mo eo e , i should be no ed ha in HC, bo h HC alone o wi h ea men (NLC+ERT o
ERT), we ound ha he a ge ed lysosome and lysosomal memb ane o ganiza ion p o ein le els
dec ease (Figu e 5A,B).
Figu e 4.
FunRich unc ional analysis esul s o exp essed p o eins om he me abolic p ocess o he
endosome. (
A
) compa ison be ween un ea ed and ea ed o 24 h wi h NLC +ERT in HC and MoC;
(B) compa ison be ween un ea ed and ea ed o 24 h wi h ERT in HC and MoC.
In he lysosomal memb ane (Figu e 5), we obse ed a dec ease o he exp ession le el in he
p o eins ela ed o a ge ing he lysosome and o he lysosomal memb ane o ganiza ion in
NLC +ERT
(Figu e 5A). Howe e , in ERT ea men s, we only ound a dec ease in he lysosomal memb ane
(Figu e 5B). Mo eo e , i should be no ed ha in HC, bo h HC alone o wi h ea men (NLC +ERT
o ERT), we ound ha he a ge ed lysosome and lysosomal memb ane o ganiza ion p o ein le els
dec ease (Figu e 5A,B).
Rela ed o he biological p ocessing o p o eins in he lysosome (Figu e 6), we analyzed bo h he
biosyn hesis and he ca abolism o C6S and KS. We obse ed ha in cells ea ed wi h NLC +ERT
(Figu e 6A) o ERT (Figu e 6B), he p o eins ela ed o hese p ocesses we e dec eased.
In . J. Mol. Sci. 2019,20, 4610 6 o 22
In . J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 6 o 22
Figu e 5. FunRich unc ional analysis esul s o exp essed p o eins om he me abolic
p ocess o he lysosomal memb ane. (A) compa ison be ween no ea men and 24 h
ea men wi h NLC + ERT in HC and MoC (B) compa ison be ween no ea men and 24 h
ea men wi h ERT in HC and MoC.
Rela ed o he biological p ocessing o p o eins in he lysosome (Figu e 6), we analyzed bo h he
biosyn hesis and he ca abolism o C6S and KS. We obse ed ha in cells ea ed wi h NLC+ERT
(Figu e 6A) o ERT (Figu e 6B), he p o eins ela ed o hese p ocesses we e dec eased.
Figu e 6. FunRich unc ional analysis esul s o he exp essed p o eins om he me abolic
p ocess o he lysosome. (A) compa ison be ween no ea men and 24 h ea men wi h
NLC + ERT in HC and MoC (B) compa ison be ween no ea men and 24 h ea men wi h
ERT in HC and MoC.
We also analyzed he biological p ocesses o p o eins in he soluble N-e hylmaleimide-sensi i e
ac o a achmen p o ein ecep o (SNARE) complex (Figu e 7), speci ically looking o changes in
he me abolic pa hway o choles e ol and SNARE complex disassembly. The pe cen age o p o eins
in ol ed in hese p ocesses was inc eased wi h bo h ea men s.
Figu e 5.
FunRich unc ional analysis esul s o exp essed p o eins om he me abolic p ocess o he
lysosomal memb ane. (
A
) compa ison be ween no ea men and 24 h ea men wi h NLC +ERT in
HC and MoC (B) compa ison be ween no ea men and 24 h ea men wi h ERT in HC and MoC.
In . J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 6 o 22
Figu e 5. FunRich unc ional analysis esul s o exp essed p o eins om he me abolic
p ocess o he lysosomal memb ane. (A) compa ison be ween no ea men and 24 h
ea men wi h NLC + ERT in HC and MoC (B) compa ison be ween no ea men and 24 h
ea men wi h ERT in HC and MoC.
Rela ed o he biological p ocessing o p o eins in he lysosome (Figu e 6), we analyzed bo h he
biosyn hesis and he ca abolism o C6S and KS. We obse ed ha in cells ea ed wi h NLC+ERT
(Figu e 6A) o ERT (Figu e 6B), he p o eins ela ed o hese p ocesses we e dec eased.
Figu e 6. FunRich unc ional analysis esul s o he exp essed p o eins om he me abolic
p ocess o he lysosome. (A) compa ison be ween no ea men and 24 h ea men wi h
NLC + ERT in HC and MoC (B) compa ison be ween no ea men and 24 h ea men wi h
ERT in HC and MoC.
We also analyzed he biological p ocesses o p o eins in he soluble N-e hylmaleimide-sensi i e
ac o a achmen p o ein ecep o (SNARE) complex (Figu e 7), speci ically looking o changes in
he me abolic pa hway o choles e ol and SNARE complex disassembly. The pe cen age o p o eins
in ol ed in hese p ocesses was inc eased wi h bo h ea men s.
Figu e 6.
FunRich unc ional analysis esul s o he exp essed p o eins om he me abolic p ocess o
he lysosome. (
A
) compa ison be ween no ea men and 24 h ea men wi h NLC +ERT in HC and
MoC (B) compa ison be ween no ea men and 24 h ea men wi h ERT in HC and MoC.
We also analyzed he biological p ocesses o p o eins in he soluble N-e hylmaleimide-sensi i e
ac o a achmen p o ein ecep o (SNARE) complex (Figu e 7), speci ically looking o changes in
he me abolic pa hway o choles e ol and SNARE complex disassembly. The pe cen age o p o eins
in ol ed in hese p ocesses was inc eased wi h bo h ea men s.
In . J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 6 o 22
Figu e 5. FunRich unc ional analysis esul s o exp essed p o eins om he me abolic
p ocess o he lysosomal memb ane. (A) compa ison be ween no ea men and 24 h
ea men wi h NLC + ERT in HC and MoC (B) compa ison be ween no ea men and 24 h
ea men wi h ERT in HC and MoC.
Rela ed o he biological p ocessing o p o eins in he lysosome (Figu e 6), we analyzed bo h he
biosyn hesis and he ca abolism o C6S and KS. We obse ed ha in cells ea ed wi h NLC + ERT
(Figu e 6A) o ERT (Figu e 6B), he p o eins ela ed o hese p ocesses we e dec eased.
Figu e 6. FunRich unc ional analysis esul s o he exp essed p o eins om he me abolic
p ocess o he lysosome. (A) compa ison be ween no ea men and 24 h ea men wi h
NLC + ERT in HC and MoC (B) compa ison be ween no ea men and 24 h ea men wi h
ERT in HC and MoC.
We also analyzed he biological p ocesses o p o eins in he soluble N-e hylmaleimide-sensi i e
ac o a achmen p o ein ecep o (SNARE) complex (Figu e 7), speci ically looking o changes in
he me abolic pa hway o choles e ol and SNARE complex disassembly. The pe cen age o p o eins
in ol ed in hese p ocesses was inc eased wi h bo h ea men s.
Figu e 7.
FunRich unc ional analysis esul s o exp essed p o eins om he me abolic p ocess
o he soluble N-e hylmaleimide-sensi i e ac o a achmen p o ein ecep o (SNARE) complex.
(
A
) compa ison be ween non- ea men and 24 h ea men wi h NLC+ERT in HC and MoC (
B
)
compa ison be ween non- ea men and 24 h ea men wi h ERT in HC and MoC.
In . J. Mol. Sci. 2019,20, 4610 7 o 22
Wi h ega ds o he GAG deg ada ion ou e (Figu e 8), ou esul s showed ha , in UMoC only
h ee o he ou p o eins implica ed in he pa hway we e p esen . Howe e , when we added NCL+ERT
o ERT (da a no shown), all he p o eins appea ed, hus comple ing he pa hway and con i ming ha
ERT p o ides an impac on he GAG deg ada ion pa hway.
In . J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 7 o 22
Figu e 7. FunRich unc ional analysis esul s o exp essed p o eins om he me abolic
p ocess o he soluble N-e hylmaleimide-sensi i e ac o a achmen p o ein ecep o
(SNARE) complex. (A) compa ison be ween non- ea men and 24 h ea men wi h NLC+
ERT in HC and MoC (B) compa ison be ween non- ea men and 24 h ea men wi h ERT
in HC and MoC.
Wi h ega ds o he GAG deg ada ion ou e (Figu e 8), ou esul s showed ha , in UMoC only
h ee o he ou p o eins implica ed in he pa hway we e p esen . Howe e , when we added
NCL+ERT o ERT (da a no shown), all he p o eins appea ed, hus comple ing he pa hway and
con i ming ha ERT p o ides an impac on he GAG deg ada ion pa hway.
Figu e 8. DAVID unc ional analysis o he glycosaminoglycan (GAG) deg ada ion
pa hway in mucopolysaccha idosis ype IVA (MPS IVA) ib oblas s wi hou ea men (A)
o NLC+ERT (B). Red s a s co espond o p o eins iden i ied by he DDA-LC-MS/MS
analysis. G een colo o he boxes: su e con idence, black solid a ows: high con idence;
black do ed a ows: mode a e con idence.
Acco dingly, we also es ed he esicles’ in e naliza ion, which was p e iously desc ibed as
mannose-6-phospha e ecep o -dependen [43,44]. Ou esul s indica e ha NLC encapsula ion o he
d ug induces in e naliza ion o esicles in a di e en way h ough endocy osis, as we ound p o eins
in ol ed in he ini ial and inal phases o endosome o ma ion ( Figu e 2; Figu e 9).
Figu e 8.
DAVID unc ional analysis o he glycosaminoglycan (GAG) deg ada ion pa hway in
mucopolysaccha idosis ype IVA (MPS IVA) ib oblas s wi hou ea men (
A
) o NLC +ERT (
B
).
Red s a s co espond o p o eins iden i ied by he DDA-LC-MS/MS analysis. G een colo o he boxes:
su e con idence, black solid a ows: high con idence; black do ed a ows: mode a e con idence.
Acco dingly, we also es ed he esicles’ in e naliza ion, which was p e iously desc ibed as
mannose-6-phospha e ecep o -dependen [
43
,
44
]. Ou esul s indica e ha NLC encapsula ion o he
d ug induces in e naliza ion o esicles in a di e en way h ough endocy osis, as we ound p o eins
in ol ed in he ini ial and inal phases o endosome o ma ion (Figu es 2and 9).
We ob ained hequan i a i emeasu emen o p o einsbyano he me hod (desc ibedinSec ion 2.2).
We analyzed he same specimens wi h a new da abase (shown in Supplemen a y Table S1). In Figu e 10,
we show he pe cen age o up egula ed p o eins, compa ing HC, UMoC, and MoC wi h NLC +ERT;
MoC wi h ERT alone; HC wi h NLC +ERT; and HC wi h ERT alone in di e en o ganelles. The p o ein
pa e n exp essions in he endolysosomal memb ane a e simila o hose in he un ea ed a ec ed
cells and he HC. Howe e , p o eins ela ed o he mi ochond ion, lysosomal memb ane, lysosome,
and endosome we e up egula ed in he HC. Likewise, 17% o p o eins in MoC ea ed wi h NLC +ERT
we e ele a ed in he mi ochond ion when compa ed wi h he un ea ed cells. No signi ican di e ence
was ound in he emaining o ganelles.
In . J. Mol. Sci. 2019,20, 4610 8 o 22
In . J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 8 o 22
Figu e 9. DAVID unc ional analysis o esicle in e naliza ion. Red s a s co espond o p o eins
iden i ied by he DDA-LC-MS/MS analysis. G een colo o he boxes: su e con idence, black solid
a ows: high con idence; black do ed a ows: mode a e con idence.
We ob ained he quan i a i e measu emen o p o eins by ano he me hod (desc ibed in Sec ion
3.2). We analyzed he same specimens wi h a new da abase (shown in Supplemen a y Table 1). In
Figu e 10, we show he pe cen age o up egula ed p o eins, compa ing HC, UMoC, and MoC wi h
NLC+ERT; MoC wi h ERT alone; HC wi h NLC+ERT; and HC wi h ERT alone in di e en o ganelles.
The p o ein pa e n exp essions in he endolysosomal memb ane a e simila o hose in he un ea ed
a ec ed cells and he HC. Howe e , p o eins ela ed o he mi ochond ion, lysosomal memb ane,
lysosome, and endosome we e up egula ed in he HC. Likewise, 17% o p o eins in MoC ea ed wi h
NLC+ERT we e ele a ed in he mi ochond ion when compa ed wi h he un ea ed cells. No
signi ican di e ence was ound in he emaining o ganelles.
Figu e 10. FunRich Func ional analysis esul s o up egula ed p o eins showing he esul s
ela ed o p o ein in e naliza ion.
2.2. P o ein Quan i ica ion by SWATH-MS
Figu e 9.
DAVID unc ional analysis o esicle in e naliza ion. Red s a s co espond o p o eins
iden i ied by he DDA-LC-MS/MS analysis. G een colo o he boxes: su e con idence, black solid
a ows: high con idence; black do ed a ows: mode a e con idence.
In . J. Mol. Sci. 2019, 20, x FOR PEER REVIEW 8 o 22
Figu e 9. DAVID unc ional analysis o esicle in e naliza ion. Red s a s co espond o p o eins
iden i ied by he DDA-LC-MS/MS analysis. G een colo o he boxes: su e con idence, black solid
a ows: high con idence; black do ed a ows: mode a e con idence.
We ob ained he quan i a i e measu emen o p o eins by ano he me hod (desc ibed in Sec ion
3.2). We analyzed he same specimens wi h a new da abase (shown in Supplemen a y Table 1). In
Figu e 10, we show he pe cen age o up egula ed p o eins, compa ing HC, UMoC, and MoC wi h
NLC+ERT; MoC wi h ERT alone; HC wi h NLC+ERT; and HC wi h ERT alone in di e en o ganelles.
The p o ein pa e n exp essions in he endolysosomal memb ane a e simila o hose in he un ea ed
a ec ed cells and he HC. Howe e , p o eins ela ed o he mi ochond ion, lysosomal memb ane,
lysosome, and endosome we e up egula ed in he HC. Likewise, 17% o p o eins in MoC ea ed wi h
NLC+ERT we e ele a ed in he mi ochond ion when compa ed wi h he un ea ed cells. No
signi ican di e ence was ound in he emaining o ganelles.
Figu e 10. FunRich Func ional analysis esul s o up egula ed p o eins showing he esul s
ela ed o p o ein in e naliza ion.
2.2. P o ein Quan i ica ion by SWATH-MS
Figu e 10.
FunRich Func ional analysis esul s o up egula ed p o eins showing he esul s ela ed o
p o ein in e naliza ion.
2.2. P o ein Quan i ica ion by SWATH-MS
We employed his app oach based on he use o a lib a y in which quan i ica ion is pe o med
o s udy de egula ed p o eins. To gene a e his lib a y, he samples we e pooled, and 4
µ
L o each
sample was loaded in o an DDA-LC-MS/MS sys em. A o al o 1019 di e en p o eins we e iden i ied
(Supplemen a y Da a 1). These p o eins we e selec ed because hey had a global alse disco e y
a e <1% and an unused sco e (p o ein sco e) o >1.2.
We ound 11 up egula ed p o eins in UMoC, compa ed in ERT ea ed cells and, con e sely, 43
up egula ed p o eins in MoC wi h ERT, compa ed in UMoC. These p o eins had a p<0.05 and a old
change (FC) >1.2 (Table 2).
In . J. Mol. Sci. 2019,20, 4610 9 o 22
Table 2. Quan i a i e s udy o p o eins in UMoC s. MoC wi h ERT (A) and ice e sa (B).
A. Up egula ed P o eins in UMoC, Compa ed in MoC wi h ERT
P o ein G oup p-Value FC
P29692 Elonga ion ac o 1-del a 0.0341 2.1053
P54727 UV excision epai p o ein RAD23 homolog B 0.0340 1.9325
P16401 His one H1.5 0.0357 1.8159
P26373 60S ibosomal p o ein L13 0.0449 1.8140
O15143 Ac in- ela ed p o ein 2/3 complex subuni 1B 0.0329 1.7815
P31942 He e ogeneous nuclea ibonucleop o ein H3 0.0234 1.6284
P07437 Tubulin be a chain 0.0361 1.6138
Q07020
Q9Y4L1 60S ibosomal p o ein L18
Hypoxia up- egula ed p o ein 1 0.0477
0.0104 1.4902
1.4313
P13674 P olyl 4-hyd oxylase subuni alpha-1 0.0299 1.3145
P68104 Elonga ion ac o 1-alpha 1 0.0342 1.2451
B. Up egula ed P o eins in MoC wi h ERT, Compa ed in UMoC
P o ein G oup p-Value FC
P07237 P o ein disul ide-isome ase 0.0408 1.2058
P78417 Glu a hione S- ans e ase omega-1 0.0425 1.2224
O00299 Chlo ide in acellula channel p o ein 1 0.0195 1.2303
P23526 Adenosylhomocys einase 0.0443 1.2358
Q96S97 Myeloid-associa ed di e en ia ion ma ke 0.0496 1.2446
P62258 14-3-3 p o ein epsilon 0.0079 1.2498
P16152 Ca bonyl educ ase [NADPH] 1 0.0199 1.2521
P32119 Pe oxi edoxin-2 0.0385 1.2540
P00387 NADH-cy och ome b5 educ ase 3 0.0087 1.2629
P17655 Calpain-2 ca aly ic subuni 0.0287 1.2708
P61981 14-3-3 P o ein gamma 0.0482 1.2743
P60900 P o easome subuni alpha ype-6 0.0270 1.2756
O43707 Alpha-ac inin-4 0.0309 1.2781
P27824 Calnexin 0.0254 1.2798
P11413 Glucose-6-phospha e 1-dehyd ogenase 0.0215 1.2906
P08133 Annexin A6 0.0055 1.2937
P12814 Alpha-ac inin-1 0.0146 1.3017
P22314 Ubiqui in-like modi ie -ac i a ing enzyme 1 0.0015 1.3163
P54578 Ubiqui in ca boxyl- e minal hyd olase 14 0.0386 1.3214
P49721 P o easome subuni be a ype-2 0.0414 1.3568
Q96AG4 Leucine- ich epea -con aining p o ein 59 0.0085 1.3642
P67812 Signal pep idase complex ca aly ic subuni SEC11A 0.0153 1.3671
Q15404 Ras supp esso p o ein 1 0.0172 1.3808
P07996 Th ombospondin-1 0.0379 1.3992
O00629 Impo in subuni alpha-3 0.0386 1.4218
Q9BS26 Endoplasmic e iculum esiden p o ein 44 0.0170 1.4754
Q99798 Aconi a e hyd a ase, mi ocond ial 0.0357 1.4799
P60953 Cell di ision con ol p o ein 42 homolog 0.0215 1.4806
Q9Y3I0 RNA-splicing ligase R cB homolog 0.0506 1.4807
P2484 Myosin egula o y ligh polypep ide 9 0.0344 1.5135
Q15758 Neu al amino acid anspo e B(0) 0.0076 1.5970
Q13724 Mannosyl-oligosaccha ide glucosidase 0.0351 1.6011
P08727 Ke a in, ype I cy oskele al 19 0.0315 1.6256
P24941 Cyclin-dependen kinase 2 0.0114 1.6367
P61619 P o ein anspo p o ein Sec61 subuni alpha iso o m 1 0.0398 1.7619
P08195 4F2 cell-su ace an igen hea y chain 0.0151 1.7870
Q16881 Thio edoxin educ ase 1, cy oplasmic 0.0472 1.8402
P55060 Expo in-2 0.0229 1.9550
P04179 Supe oxide dismu ase [Mn], mi ocond ial 0.0066 2.3668
Q9NVD7 Alpha-pa in 0.0339 2.3802
Q9HDC9 Adipocy e plasma memb ane-associa ed p o ein 0.0129 2.4386
In . J. Mol. Sci. 2019,20, 4610 16 o 22
Table 6. Values o enzyme ac i i y.
Samples Be o e
T ea men (nM/h/mg)
A e
T ea men (nM/h/mg)
S.1 MoC 0.0 1.6
S.2 MoC 0.0 4.8
S.3 MoC 0.0 6.5
S.4 MoC 0.0 8.9
4.7. P o eomic Analysis
4.7.1. In Gel P o ein Diges ion
To ensu e global and quan i a i e (by SWATH-MS) p o ein iden i ica ion, an equal amoun o
p o eins om he ea ed pa ien s and con ols we e loaded on a 10% SDS-PAGE gel. The un was
s opped as soon as he on had pene a ed 3 mm in o he esol ing gel [
59
,
60
]. The p o ein band
was de ec ed by Syp o-Ruby luo escen s aining (Lonza, Basel, Swi ze land), excised, and p ocessed
o in-gel and manual yp ic diges ion, as desc ibed [
61
]. Gel pieces we e educed wi h 10 mM
di hio h ei ol (Sigma-Ald ich, S . Louis, MO, USA) in 50 mM ammonium bica bona e (Sigma-Ald ich,
S . Louis, MO, USA) and alkyla ed wi h 55 mM iodoace amide (Sigma-Ald ich, S . Louis, MO, USA) in
50 mM ammonium bica bona e. Then, he gel pieces we e insed wi h 50 mM ammonium bica bona e
in 50% me hanol (HPLC g ade, Scha lau, Ba celona, Spain), dehyd a ed ia he addi ion o ace oni ile
(HPLC g ade, Scha lau, Ba celona, Spain), and d ied in a SpeedVac. Modi ied po cine ypsin (P omega,
Madison, WI, USA) was added o he d y gel pieces a a inal concen a ion o 20 ng/
µ
L in 20 mM
ammonium bica bona e, incuba ing hem a 37
◦
C o 16 h. The pep ides we e ex ac ed h ice by
20 min incuba ion in 40
µ
L o 60% ace oni ile in 0.5% o mic acid. The esul ing pep ide ex ac s
we e pooled, concen a ed in a SpeedVac (Te moFishe Scien i ic: Haysham, Lancashi e), and s o ed a
−20 ◦C.
4.7.2. Mass Spec ome ic Analysis (DDA acquisi ion)
Diges ed pep ides we e sepa a ed using Re e se Phase Ch oma og aphy. The g adien was
c ea ed using a mic o liquid ch oma og aphy sys em (Eksigen Technologies nanoLC 400, ABSciex,
(Wa ing on, Cheshi e, UK) coupled o high-speed T iple TOF 6600 mass spec ome e (ABSciex,
Fos e Ci y, CA, USA), wi h a mic o low sou ce, as desc ibed p e iously [
26
,
27
]. The chosen analy ical
column was a silica-based e e sed-phase column Eksigen C18CL 150
×
0.30 mm, 3
µ
m pa icle size,
and 120 Å po e size (Eksigen , ABSciex, Woodlands Cen al Indus. Es a e, Singapo e). The ap column
was a YMC-TRIART C18 (YMC Technologies, Teknok oma, Ba celona, Spain) wi h a 3
µ
m pa icle
size and 120 Å po e size, swi ched on-line wi h he analy ical column. The loading pump deli e ed a
solu ion o 0.1% o mic acid in wa e a 10
µ
L/min. The mic o-pump gene a ed a low- a e o 5
µ
L/min
and ope a ed unde g adien elu ion condi ions, using 0.1% o mic acid in he wa e as mobile phase
A, and 0.1% o mic acid in ace oni ile as mobile phase B. The pep ides we e sepa a ed using a 90 min
g adien anging om 2% o 90% o mobile phase B (mobile phase A: 2% ace oni ile, 0.1% o mic acid;
mobile phase B: 100% ace oni ile, 0.1% o mic acid). The injec ion olume was 4
µ
L (mo e o less 4
µ
g
o p o ein).
Da a acquisi ion was pe o med in a T ipleTOF 6600 Sys em (ABSciex, Fos e Ci y, CA, USA)
using a da a dependen wo k low. Sou ce and in e ace condi ions we e he ollowing: ion sp ay
ol age loa ing (ISVF) 5500 V, cu ain gas (CUR) 25, collision ene gy (CE) 10, and ion sou ce gas 1
(GS1) 25. The ins umen s we e ope a ed wi h he Analys TF 1.7.1 so wa e (ABSciex, Woodlands
Cen al Indus. Es a e, Singapo e). The swi ching c i e ia we e se o ions g ea e han he mass o
cha ge a io (m/z) 350 and smalle han m/z1400, wi h a cha ge s a e o 2–5, a mass ole ance o 250 ppm,
and an abundance h eshold o mo e han 200 coun s (cps). Fo me a ge ions we e excluded o 15 s.
In . J. Mol. Sci. 2019,20, 4610 17 o 22
The ins umen s we e au oma ically calib a ed e e y 4 h using ex e nal calib an yp ic pep ides om
PepCalMix (Sciex, Wa ing on, Cheshi e, UK).
4.7.3. Da a Analysis
A e he MS/MS analysis, he da a iles we e p ocessed using he P o einPilo TM 5.0.1 so wa e
om ABSciex (Woodlands Cen al Indus Es a e, Singapo e), which uses he algo i hm Pa agonTM o
da abase sea ching and P og oupTM o da a g ouping. Da a we e sea ched using a Human-speci ic
Unip o da abase (www.unip o .o g). The alse disco e y a e was de e mined using a non-linea
i ing me hod displaying only hose esul s ha epo ed a 1% Global alse disco e y a e o be e [
28
].
Func ionalanalysiswaspe o medbydi e en openaccessso wa e. Weused
FunRich
( unc ional
en ichmen analysis ool) o unc ional en ichmen and in e ac ion ne wo k analysis (A ailable online:
h p:// un ich.o g/index.h ml). Fo s a is ics, we used FunRich (hype geome ic es ), Bon e oni [
29
,
30
],
ne wo k cons uc ion, and clus e ing. We also used DAVID (A ailable online: h ps://da id.nci c .go /)
o GAG deg ada ion pa hways and unc ional analysis o he esicle in e naliza ion.
4.8. P o ein Quan i ica ion by SWATH-MS
4.8.1. C ea ion o he Spec al Lib a y
To build he MS/MS spec al lib a ies, he pep ide solu ions we e analyzed by a sho gun
da a-dependen acquisi ion (DDA) app oach using mic o-LC-MS/MS (ABSciex, Redwood Ci y, CA,
USA), as desc ibed p e iously by us and o he au ho s [27,46,47,62]. To ob ain a good ep esen a ion
o he pep ides and p o eins p esen in all samples, pooled ials o he samples om each g oup
we e p epa ed using equal mix u es o he o iginal samples. A o al o 4
µ
L om each pool was
sepa a ed in o a mic o-LC sys em Ekspe nLC425 (Eksigen, Dublin, CA, USA) using an Eksigen
C18
150 ×0.30 mm
, wi h 3 mm pa icle size and 120 Å po e size (Eksigen , ABSciex), a a low a e
o 5 mL/min. Wa e and ACN, bo h con aining 0.1% o mic acid, we e used as sol en s A and B,
espec i ely. The g adien un consis ed o 5% o 95% B o 30 min, 5 min a 90% B, and, inally, 5 min
a 5% B o column equilib a ion, o a o al un ime o 40 min. As he pep ides elu ed, hey we e
di ec ly injec ed in o a hyb id quad upole-TOF mass spec ome e T iple TOF 6600 (ABSciex, Redwood
Ci y, CA, USA) ope a ed wi h a da a-dependen acquisi ion sys em in posi i e ion mode. A Mic o
sou ce (ABSciex) was used o he in e ace be ween mic oLC and MS, wi h an applica ion o 2600 V
ol age. The acquisi ion mode consis ed o a 250 ms su ey MS scan om 400 o 1250 m/z, ollowed
by an MS/MS scan om 100 o 1500 m/z(25 ms acquisi ion ime) o he op 65 p ecu so ions om
he su ey scan, o a o al cycle ime o 2.8 s. The agmen ed p ecu so s we e added o a dynamic
exclusion lis o 15 s. Any singly cha ged ions we e excluded om he MS/MS analysis.
The pep ide and p o ein iden i ica ions we e pe o med using P o ein Pilo so wa e ( e sion
5.0.1, ABSciex), and he da a we e sea ched using a Human-speci ic Unip o da abase, speci ying
iodoace amide as he Cys alkyla ion. The alse disco e y a e (FDR) was se o 1 o bo h pep ides
and p o eins. The MS/MS spec a o he iden i ied pep ides we e hen used o gene a e he spec al
lib a y o he SWATH-MS peak ex ac ion using he add-in o he PeakView So wa e ( e sion 2.2,
ABSciex) MS/MSALL wi h he SWATH-MS Acquisi ion Mic oApp ( e sion 2.0, ABSciex). Pep ides
wi h a con idence sco e abo e 99% (as ob ained om P o ein Pilo da abase sea ch) we e included in
he spec al lib a y).
4.8.2. Rela i e Quan i ica ion by SWATH-MS Acquisi ion
SWATH–MS acquisi ion was pe o med on a T ipleTOF
®
6600 LC-MS/MS sys em (ABSciex). Each
sample (4
µ
L) was analyzed using he LC-MS/MS equipmen (Sciex, Wa ing on, Cheshi e, UK) and
he LC g adien desc ibed abo e o building he spec al lib a y, bu ins ead used he SWATH-MS
acquisi ion me hod. The me hod consis ed o epea ing a cycle ha consis ed o he acquisi ion o
65 TOF MS/MS scans (400 o 1500 m/z, high sensi i i y mode, 50 ms acquisi ion ime) o o e lapping
In . J. Mol. Sci. 2019,20, 4610 18 o 22
sequen ial p ecu so isola ion windows o a iable wid hs (1 m/zo e lap), co e ing he 400 o 1250
m/zmass ange wi h a p e ious TOF MS scan (400 o 1500 m/z, 50 ms acquisi ion ime) o each cycle.
The o al cycle ime was 6.3 s. Fo each sample se , he wid h o he 65 a iable windows was op imized
acco ding o he ion densi y ound in he DDA uns using a SWATH-MS a iable window calcula o
wo kshee om Sciex.
4.8.3. Da a Analysis
The a ge ed da a ex ac ion o he agmen ion ch oma og am aces om he SWATH-MS uns
was pe o med by Peak View ( e sion 2.2, ABSciex) using he SWATH-MS Acquisi ion Mic o App
( e sion 2.0, Sciex, Wa ing on, Cheshi e, UK). This applica ion p ocessed he da a using he spec al
lib a y c ea ed om he sho gun da a. Up o en pep ides pe p o ein and se en agmen s pe pep ide
we e selec ed, based on signal in ensi y. Any sha ed and modi ied pep ides we e excluded om he
p ocessing. Fi e-minu e windows and 30 ppm wid hs we e used o ex ac he ion ch oma og ams.
SWATH-MS quan iza ion was a emp ed o all p o eins in he ion lib a y ha we e iden i ied by
P o ein Pilo wi h an FDR below 1%. The e en ion imes om he pep ides ha we e selec ed o
each p o ein we e ealigned in each un, acco ding o he iRT pep ides ha spiked in each sample,
and elu ed along he whole- ime axis. The ex ac ed ion ch oma og ams we e hen gene a ed o each
selec ed agmen ion. The peak a eas o he pep ides we e ob ained by summing he peak a eas om
he co esponding agmen ions. Peak View compu ed an FDR and a sco e o each assigned pep ide
acco ding o he ch oma og aphic and spec a componen s. Only pep ides wi h an FDR below 5%
we e used o p o ein quan iza ion. P o ein quan iza ion was calcula ed by adding he peak a eas o
he co esponding pep ides.
The in eg a ed peak a eas we e di ec ly expo ed o he Ma ke View so wa e (ABSciex) o
ela i e quan i a i e analysis. The expo gene a ed h ee iles con aining quan i a i e in o ma ion
abou indi idual ions, he summed in ensi y o he di e en ions o a pa icula pep ide, and he
summed in ensi y o di e en pep ides o a pa icula p o ein. Ma ke View was used o analysis o
he SWATH-MS da a epo ed in o he p o eomics s udies [
34
,
62
–
64
] because o i s da a-independen
me hod o quan iza ion. Ma ke View uses p ocessing algo i hms ha accu a ely ind ch oma og aphic
and spec al peaks di ec om he aw SWATH-MS da a. Da a alignmen by Ma ke View compensa ed
o mino a ia ions in bo h he mass and e en ion ime alues, ensu ing ha iden ical compounds in
di e en samples we e accu a ely compa ed o one ano he . To con ol o possible une en sample
loss ac oss he di e en samples du ing he sample p epa a ion p ocess, we pe o med a global
no maliza ion based on he o al sum o all he peak a eas ex ac ed om all he pep ides and
ansi ions ac oss he eplica es o each sample [
65
]. An unsupe ised mul i a ia e s a is ical analysis
using p incipal componen analysis (PCA) was pe o med o compa e he da a ac oss he samples.
The a e age MS peak a ea o each p o ein was de i ed om he eplica es o he SWATH-MS o each
sample ollowed by a S uden ’s - es analysis using he Ma ke View so wa e o compa ison among
he samples based on he a e aged a ea sums o all he ansi ions de i ed o each p o ein. The - es
indica ed how well each a iable dis inguished he wo g oups, epo ed as a p- alue. Fo each lib a y,
each se o di e en ially exp essed p o eins (p- alue <0.05) wi h 1.2 up- egula ed o down- egula ed
p o eins was selec ed.
5. Conclusions
We ha e demons a ed ha ou new deli e y sys em, h ough a nanos uc u e lipid ca ie , can
induce changes in p o eins ha a e in ol ed in he disease. This sys em used a di e en in e naliza ion
pa hway (no sa u able) ha seems o induce be e access o he lysosome. Using a low enzyme
dosage, we ob ained excellen C6S and KS deg ada ion, g ea e han wi h he ee enzyme. The e o e,
his sys em can imp o e he lysosome-endosome-mi ochond ia machine in he MoC.
Supplemen a y Ma e ials:
Supplemen a y ma e ialscan be ound a h p://www.mdpi.com/1422-0067/20/18/4610/s1.
In . J. Mol. Sci. 2019,20, 4610 19 o 22
Au ho Con ibu ions:
Con ibu ions we e p o ided by he au ho s in he s a emen s: concep ualiza ion, J.V.
Á
.,
S.B.B., F.J.O.-E., and M.L.C.; me hodology, S.B.B., J.V.
Á
., M.G.-V.; alida ion, J.V.
Á
., and S.B.B.; o mal analysis,
J.V.
Á
., S.B.B. and C.C.; w i ing—o iginal d a p epa a ion, J.V.
Á
., S.B.B.; w i ing— e iew and edi ing, M.L.C.,
M.J.D.C, F.J.O.-E., A.L., and S.T.; isualiza ion, S.B.B.; supe ision, M.L.C., F.J.O.-E., A.L., and S.T.
Con lic s o In e es :
J. V
í
c o
Á
l a ez, As e ia Luza do, C is
ó
bal Col
ó
n, F ancisco J. O e o-Espina , and Ma
í
a L.
Couce decla e compe ing inancial in e es s: They ha e a pa en applica ion, PCT/EP2019/068629. This pa en
applica ion co e s he unde lying concep o immobilized ERT on nanos uc u ed lipid sys ems o ea MPS IVA,
as desc ibed in he manusc ip .
Abb e ia ions
C6S Chond oi in 6 sulpha e
ERT Enzyme eplacemen he apy
FC Fold change
GAG Glycosaminoglycan
GALNS N-ace ylgalac osamine-6-sul a ase
KS Ke a an sulpha eJ
LC Liquid ch oma og aphy
MPS IVA Mucopolysaccha idosis ype IVA
MRM Mul iple eac ion moni o ing
MS Mass spec ome y
NLC Nanos uc u e lipid ca ie
S Sample
SDC Sodium dodecyl sul a e
SRM Selec ed eac ion moni o ing
SNARE soluble N-e hylmaleimide-sensi i e ac o a achmen p o ein ecep o
SWATH-MS Sequen ial window acquisi ion o all heo e ical mass spec a
UMoC Un ea ed Mo quio A cells
Re e ences
1.
Hend iksz, C.J.; Be ge , K.I.; Giugliani, R.; Ha ma z, P.; Kampmann, C.; Mackenzie, W.G.; Raiman, J.;
Villa eal, M.S.; Sa a i ayan, R. In e na ional Guidelines o he Managemen and T ea men o Mo quio A
Synd ome. Am. J. Med. Gene . A 2015,167, 11–25. [C ossRe ] [PubMed]
2.
Khan, S.; Alm
é
ciga-D
í
az, C.J.; Sawamo o, K.; Mackenzie, W.G.; The oux, M.C.; Piza o, C.; Mason, R.W.;
O ii, T.; Toma su, S. Mucopolysaccha idosis IVA and glycosaminoglycans. Mol. Gene . Me ab.
2017
,
120, 78–95. [C ossRe ]
3.
Mon año, A.M.; Toma su, S.; Go esman, G.S.; Smi h, M.; O ii, T. In e na ional Mo quioA Regis y: Clinical
mani es a ion and na u al cou se o Mo quio A disease. J. Inhe i . Me ab. Dis.
2007
,30, 165–174. [C ossRe ]
[PubMed]
4.
Toma su, S.; Mon año, A.M.; Oikawa, H.; Smi h, M.; Ba e a, L.; Chinen, Y.; Thacke , M.M.; Mackenzie, W.G.;
Suzuki, Y.; O ii, T. Mucopolysaccha idosis ype IVA (Mo quio A disease): Clinical e iew and cu en
ea men . Cu . Pha m. Bio echnol. 2011,12, 931–945. [C ossRe ]
5. La e y, C.; Hend iksz, C. Mo ali y in pa ien s wi h Mo quio Synd ome A. JIMD Rep. 2015,15, 59–66.
6.
Hend iksz, C.J. Elosul aseal a (BMN 110) o he ea men o mucopolysaccha idosis IVA (Mo quio A
Synd ome). Expe Re . Clin. Pha m. 2016,9, 1521–1532. [C ossRe ] [PubMed]
7.
Hend iksz,C.; San a,S.; Jones,S.A.; Gebe hiwo ,T.; Jesai is, L.; Long,B.; Qi, Y.; Hawley,S.M.; Decke ,C.Sa e y,
immunogenici y, and clinical ou comes in pa ien s wi h Mo quio A synd ome pa icipa ing in 2 sequen ial
open-label s udies o elosul ase al a enzyme eplacemen he apy (MOR-002/MOR-100), ep esen ing 5 yea s
o ea men . Mol. Gene . Me ab. 2018,123, 479–487. [C ossRe ] [PubMed]
8.
Dohe y, C.; S aple on, M.; Piechnik, M.; Mason, R.W.; Mackenzie, W.G.; Yamaguchi, S.; Kobayashi, H.;
Suzuki, Y.; Toma su, S. E ec o enzyme eplacemen he apy on he g ow h o pa ien s wi h Mo quio A.
J. Hum. Gene . 2019,64, 625–635. [C ossRe ]
In . J. Mol. Sci. 2019,20, 4610 20 o 22
9.
Toma su, S.; A e ill, L.W.; Sawamo o, K.; Mackenzie, W.G.; Bobe , M.B.; Piza o, C.; Go , C.J.; Xie, L.;
O ii, T.; The oux, M. Obs uc i e ai way in Mo quio A synd ome, he pas , he p esen and he u u e.
Mol. Gene . Me ab. 2016,117, 150–156. [C ossRe ]
10.
Toma su, S.; Sawamo o, K.; Shimada, T.; Bobe , M.B.; Kubaski, F.; Yasuda, E.; Mason, R.W.; Khan, S.;
Alm
é
ciga-D
í
az, C.J.; Ba e a, L.A.; e al. Enzyme eplacemen he apy o ea ing mucopolysaccha idosis
ype IVA (Mo quio A synd ome): E ec and limi a ions. Expe Opin. O phan D ugs
2015
,3, 1279–1290.
[C ossRe ]
11.
Toma su, S.; Sawamo o,K.; Alm
é
ciga-D
í
az,C.J.; Shimada,T.; Bobe , M.B.; Chinen,Y.; Yabe,H.; Mon año,A.M.;
Giugliani, R.; Kubaski, F.; e al. Impac o enzyme eplacemen he apy and hema opoie ic s em cell
ansplan a ion in pa ien s wi h Mo quio A synd ome. D ug Des. De el.
2015
,9, 1937–1953. [C ossRe ]
[PubMed]
12.
Qi, Y.; Musson, D.G.; Schweigha d , B.; Tompkins, T.; Jesai is, L.; Shaywi z, A.J.; Yang, K.; O’Neill, C.A.
Pha macokine ic and pha macodynamic e alua ion o Elosul ase Al a, an enzyme eplacemen he apy in
pa ien s wi h Mo quioA Synd ome. Clin. Pha m. 2014,53, 1137–1147. [C ossRe ] [PubMed]
13.
Ken h, J.J.; Thompson, G.; Fullwood, C.; Wilkinson, S.; Jones, S.; B uce, I.A. The cha ac e isa ion o pulmona y
unc ion in pa ien s wi h mucopolysaccha idoses IVA: A longi udinal analysis. Mol. Gene . Me ab. Rep.
2019
,
20, 100487. [C ossRe ] [PubMed]
14.
Toma su, S.; Mon año, A.M.; Dung, V.C.; Ohashi, A.; Oikawa, H.; Oguma, T.; O ii, T.; Ba e a, L.; Sly, W.S.
Enhancemen o D ug Deli e y: Enzyme- eplacemen he apy o mu ine Mo quio A Synd ome. Mol. The .
2010,18, 1094–1102. [C ossRe ] [PubMed]
15.
Long, B.; Tompkins, T.; Decke , C.; Jesai is, L.; Khan, S.; Slaso , P.; Ha ma z, P.; O’Neill, C.A.; Schweigha d , B.
Long- e m Immunogenici y o Elosul ase Al a in he T ea men o Mo quioA Synd ome: Resul s F om
MOR-005, a Phase III Ex ension S udy. Clin. The . 2017,39, 118–129. [C ossRe ]
16.
Sawamo o, K.; S aple on, M.; Alm
é
ciga-D
í
az, C.J.; Espejo-Mojica, A.J.; Losada, J.C.; Sua ez, D.A.; Toma su, S.
The apeu icop ions o Mucopolysaccha idoses: Cu en andeme ging ea men s. D ugs
2019
,79,1103–1134.
[C ossRe ] [PubMed]
17.
La ille e, D.; Russell, S.J.; Cosse , F.L. Re a ge ing gene deli e y using su ace-enginee ed e o i al ec o
pa icles. Cu . Opin. Bio echnol. 2001,12, 461–466. [C ossRe ]
18.
S Geo ge, J.A. Gene he apy p og ess and p ospec s: Adeno i al ec o s. Gene
2003
,10, 1135–1141.
[C ossRe ]
19.
Buning, H.; Ried, M.U.; Pe abo, L.; Ge ne , F.M.; Hu ne , N.A.; Enssle, J.; Hallek, M. Recep o a ge ing o
adeno-associa ed i us ec o s. Gene 2003,10, 1142–1151.
20.
Loile , S.A.; Conlon, T.J.; Song, S.; Tang, Q.; Wa ing on, K.H.; Aga wal, A.; Kap u czak, M.; Li, C.; Rico di, C.;
A kinson, M.A.; e al. Ta ge ing ecombinan adeno-associa ed i us ec o s o enhance gene ans e o
panc ea ic isle s and li e . Gene 2003,10, 1551–1558. [C ossRe ]
21.
Choi, V.W.; McCa y, D.M.; Samulski, R.J. AAV hyb id se o ypes: Imp o ed ec o s o gene deli e y.
Cu . Gene 2005,5, 299–310. [C ossRe ]
22. Rabilloud, T.; Che alle , M.; Luche, S.; Lelong, C. Two-dimensional gel elec opho esis in p o eomics: Pas ,
p esen and u u e. J. P o eom. 2010,73, 2064–2077. [C ossRe ] [PubMed]
23.
Kapphahn, R.J.; Richa ds, M.J.; Fe ing on, D.A.; Fliesle , S.J. Lipid-de i ed and o he oxida i e modi ica ions
o e inal p o eins in a a model o Smi h-Lemli-Opi z synd ome. Exp. Eye Res.
2019
,178, 247–254. [C ossRe ]
[PubMed]
24.
San ana, A.M.; Thomas, F.C.; Sil a, D.G.; McCulloch, E.; Vidal, A.M.C.; Bu chmo e, R.J.S.; Faglia i, J.J.;
Ecke sall, P.D. Re e ence 1D and 2D elec opho esis maps o po en ial disease ela ed p o eins in milk whey
om lac a ing bu aloes and blood se um om bu alo cal es (Wa e bu alo, Bubalusbubalis). Res. Ve . Sci.
2018,118, 449–465. [C ossRe ] [PubMed]
25.
Vido a, V.; Spacil, Z. A e iew on mass spec ome y-based quan i a i e p o eomics: Ta ge ed and da a
independen acquisi ion. Anal. Chim. Ac a 2017,964, 7–23. [C ossRe ] [PubMed]
26.
Couselo-Seijas, M.; L
ó
pez-Canoa, J.N.; Ag a-Be mejo, R.M.; D
í
az-Rod iguez, E.; Fe nandez, A.L.;
Ma inez-Ce eijo, J.M.; Du
á
n-Muñoz, D.; B a o, S.B.; Velo, A.; Gonz
á
lez-Melcho , L.; e al. Choline gic
ac i i y egula es he sec e ome o epica dial adipose issue: Associa ion wi h a ial ib illa ion. J. Cell. Physiol.
2019,234, 10512–11522. [C ossRe ] [PubMed]
In . J. Mol. Sci. 2019,20, 4610 21 o 22
27.
Del Pila Chan ada-V
á
zquez, M.; L
ó
pez, A.C.; B a o, S.B.; V
á
zquez-Es
é
ez, S.; Acea-Neb il, B.; N
ú
ñez, C.
P o eomic analysis o he bio-co ona o med on he su ace o (Au, Ag, P )-nanopa icles in human se um.
Colloids Su . B Bioin e aces 2019,177, 141–148. [C ossRe ]
28.
Shilo , I.V.; Seymou , S.L.; Pa el, A.A.; Loboda, A.; Tang, W.H.; Kea ing, S.P.; Hun e , C.L.; Nuwaysi , L.M.;
Schae e , D.A. The Pa agon Algo i hm, a Nex Gene a ion Sea ch Engine Tha Uses Sequence Tempe a u e
Values and Fea u e P obabili ies o Iden i y Pep ides om Tandem Mass Spec a. Mol. Cell. P o eom.
2007
,
6, 1638–1655. [C ossRe ]
29.
Pa han, M.; Kee hikuma , S.; Ang, C.S.; Gangoda, L.; Quek, C.Y.J.; Williamson, N.A.; Mou ado , D.;
Siebe , O.M.; Simpson, R.J.; Salim, A.; e al. FunRich: An open access s andalone unc ional en ichmen and
in e ac ion ne wo k analysis ool. P o eomics 2015,15, 2597–2601. [C ossRe ]
30.
Pa han, M.; Kee hikuma , S.; Chisanga, D.; Alessand o, R.; Ang, C.-S.; Askenase, P.; Ba ago , A.O.;
Beni o-Ma in, A.; Camussi, G.; Clay on, A.; e al. A no el communi y d i en so wa e o unc ional
en ichmen analysis o ex acellula esicles da a. J. Ex acell. Vesicles 2017,6, 1321455. [C ossRe ]
31.
Huang, D.W.; She man, B.T.; Lempicki, R.A. Bioin o ma ics en ichmen ools: Pa hs owa d he
comp ehensi e unc ional analysis o la ge gene lis s. Nucleic Acids Res. 2009,37, 1–13. [C ossRe ]
32.
Ba allob e-Ba ei o, J.; Chung, Y.L.; May , M. P o eomics and Me abolomics o Mechanis ic Insigh s and
Bioma ke Disco e y in Ca dio ascula Disease. Re . Esp. Ca diol. (Engl. Ed.)
2013
,66, 657–661. [C ossRe ]
33.
G ego ich, Z.R.; Ge, Y. Top-down p o eomics in heal h and disease: Challenges and oppo uni ies. P o eomics
2014,14, 1195–1210. [C ossRe ]
34.
Tan, H.T.; Chung, M.C.M. Label-F ee quan i a i e phosphop o eomics e eals egula ion o
asodila o -s imula ed hosphop o ein upon S a hmin-1 Silencing in a pai o isogenic colo ec al cance cell
lines. P o eomics 2018,18, 1–30. [C ossRe ]
35.
Jayabalan, N.; Lai, A.; Nai , S.; Guanzon, D.; Scholz-Rome o, K.; Palma, P.; McIn y e, H.D.; Lappas, M.;
Salomon, C. Quan i a i e p o eomics by SWATH-MS sugges an associa ion be ween ci cula ing exosomes
and ma e nal me abolic changes in ges a ional diabe es melli us. P o eomics 2019,19, e1800164. [C ossRe ]
36.
B accia, C.; Espinal, M.P.; Pini, M.; De Pie i Tonelli, D.; A mi o i, A. A new SWATH ion lib a y o mouse
adul hippocampal neu al s em cells. Da a B . 2018,18, 1–8. [C ossRe ] [PubMed]
37.
Jamwal, R.; Ba lock, B.J.; Adusumalli, S.; Ogasawa a, K.; Simons, B.L.; Akhlaghi, F. Mul iplex and label-F ee
ela i e quan i ica ion app oach o s udying p o ein abundance o d ug me abolizing enzymes in human
li e mic osomes using SWATH-MS. J. P o eome Res. 2017,16, 4134–4143. [C ossRe ]
38.
Zhu, Y.; Zhu, J.; Lu, C.; Zhang, Q.; Xie, W.; Sun, P.; Dong, X.; Yue, L.; Sun, Y.; Yi, X.; e al. Iden i ica ion o
p o ein abundance changes in Hepa ocellula Ca cinoma Tissues using PCT–SWATH. P o eom. Clin. Appl.
2019,13, e1700179. [C ossRe ]
39.
Li, H.; Mao, Y.; Xiong, Y.; Zhao, H.H.; Shen, F.; Gao, X.; Yang, P.; Liu, X.; Fu, D. A Comp ehensi e p o eome
analysis o pe iphe al blood mononuclea cells (PBMCs) o iden i y candida e bioma ke s o Panc ea ic
Cance . Cance Genom. P o eom. 2019,16, 81–89. [C ossRe ]
40.
Chang, R.Y.K.; E he idge, N.; Nouwens, A.S.; Dodd, P.R. SWATH analysis o he synap ic p o eome in
Alzheime ’s disease. Neu ochem. In . 2015,87, 1–12. [C ossRe ]
41.
Ebha d , H.A.; Degen, S.; Tadini, V.; Schilb, A.; Johns, N.; G eig, C.A.; Fea on, K.C.H.; Aebe sold, R.; Jacobi, C.
Comp ehensi e p o eome analysis o human skele al muscle in cachexia and sa copenia: A pilo s udy.
J. Cachexia Sa copenia Muscle 2017,8, 567–582. [C ossRe ] [PubMed]
42.
Zhang, X.; Walsh, T.; A he on, J.J.; Kos ne , K.; Schulz, B.; Punyadee a, C. Iden i ica ion and alida ion o a
sali a y p o ein panel o de ec hea ailu e ea ly. The anos ics 2017,7, 4350–4358. [C ossRe ] [PubMed]
43.
Cou inho, M.F.; P a a, M.J.; Al es, S. Mannose-6-phospha e pa hway: A e iew on i s ole in lysosomal
unc ion and dys unc ion. Mol. Gene . Me ab. 2012,105, 542–550. [C ossRe ] [PubMed]
44.
Ga y-Bobo, M.; Ni d
é
, P.; Jeanjean, A.; Mo
è
e, A.; Ga cia, M. Mannose 6-phospha e ecep o a ge ing and
i s applica ions in human diseases. Cu . Med. Chem. 2007,14, 2945–2953. [C ossRe ] [PubMed]
45.
Slea , D.E.; Ding, L.; Wang, S.; Zhao, C.; Wang, Y.; Xin, W.; Zheng, H.; Moo e, D.F.; Sims, K.B.; Lobel, P. Mass
spec ome y-based p o ein p o iling o de e mine he cause o lysosomal s o age diseases o unknown
e iology. Mol. Cell. P o eom. 2009,8, 1708–1718. [C ossRe ] [PubMed]
46.
O ea, I.; Rod
í
guez-A iza, A.; Chicano-G
á
l ez, E.; A enas Vacas, M.S.; Ju ado G
á
mez, B. Disco e y o
po en ial p o ein bioma ke s o lung adenoca cinoma in b onchoal eola la age luid by SWATH MS
da a-independen acquisi ion and a ge ed da a ex ac ion. J. P o eom. 2016,138, 106–114. [C ossRe ]
In . J. Mol. Sci. 2019,20, 4610 22 o 22
47.
Ga ido-Rod
í
guez, M.; O ea, I.; Calzado, M.A.; Muñoz, E.; Ga c
í
a, V. SWATH p o eomic p o iling o
p os a e cance cells iden i ies NUSAP1 as a po en ial molecula a ge o Galiellalac one. J. P o eom. 2019,
193, 217–229. [C ossRe ]
48. Appelq is , H.; Was e , P.; Kagedal, K.; Ollinge , K. The lysosome: F om was e bag o po en ial he apeu ic
a ge . J. Mol. Cell Biol. 2013,5, 214–226. [C ossRe ]
49. Ballabio, A. The awesome lysosome. EMBO Mol. Med. 2016,8, 73–76. [C ossRe ]
50.
Todka , K.; Ilama hi, H.S.; Ge main, M. Mi ochond ia and Lysosomes: Disco e ing Bonds. F on . Cell
De . Biol. 2017,5, 106. [C ossRe ]
51.
Das, A.; Nag, S.; Mason, A.B.; Ba oso, M.M. Endosome mi ocond ia in e ac ions a e modula ed by i on
elease om ans e in. J. Cell Biol. 2016,214, 831–845. [C ossRe ] [PubMed]
52.
Liebe man, A.P.; Pue ollano, R.; Raben, N.; Slaugenhaup , S.; Walkley, S.U.; Ballabio, A. Au ophagy in
lysosomal s o age diso de s. Au ophagy 2012,8, 719–730. [C ossRe ] [PubMed]
53.
Luzio, J.P.; G ay, S.R.; B igh , N.A. Endosome-lysosome usi
ó
n. Biochem. Soc. T ans.
2010
,38, 1413–1416.
[C ossRe ] [PubMed]
54.
F aldi, A.; Annunzia a, F.; Lomba di, A.; Kaise , H.-J.; Medina, D.L.; Spampana o, C.; Fedele, A.O.;
Polishchuk, R.; So en ino, N.C.; Simons, K.; e al. Lysosomal usion and SNARE unc ion a e impai ed by
choles e ol accumula ion in lysosomal s o age diso de s. EMBO J.
2010
,29, 3607–3620. [C ossRe ] [PubMed]
55.
Han, J.; Pluhacko a, K.; Böckmann, R.A. The mul i ace ed ole o SNARE p o eins in memb ane usion.
F on . Physiol. 2017,8, 5. [C ossRe ] [PubMed]
56.
Liu, R.; Liu, Q.; Wang, K.; Dang, X.; Zhang, F. Compa a i e analysis o gene exp ession p o iles in no mal hip
human ca ilage and ca ilage om pa ien s wi h nec osis o he emo al head. A h i is Res.
2016
,18, 98.
[C ossRe ] [PubMed]
57.
Lamand
é
, S.R.; Ba eman, J.F.; Hu chison, W.; McKinlay Ga dne , R.J.; Bowe , S.P.; By ne, E.; Dahl, H.H.
Reduced collagen VI causes Be hlem myopa hy: A he e ozygous COL6A1 nonsense mu a ion esul s in
mRNA decay and unc ional haploinsu iciency. Hum. Mol. Gene . 1998,7, 981–989. [C ossRe ]
58.
Camelie , M.V.; Bu in, M.G.; De Ma i, J.; Viei a, T.A.; Ma asca, G.; Giugliani, R. P ac ical and eliable enzyme
es o he de ec ion o mucopolysaccha idosis IVA (Mo quio Synd ome ype A) in d ied blood samples.
Clin. Chim. Ac a 2011,412, 1805–1808. [C ossRe ]
59.
Bonzon-Kulichenko,E.; P
é
ez-He n
á
ndez,D.; N
ú
ñez,E.; Ma
í
nez-Acedo,P.; Na a o,P.; T e isan-He az,M.;
del Ca men Ramos, M.; Sie a, S.; Ma
í
nez-Ma
í
nez, S.; Ruiz-Meana, M.; e al. A obus me hod
o quan i a i e high- h oughpu analysis o p o eomes by 18O labeling. Mol. Cell. P o eom.
2011
,
10, M110.003335. [C ossRe ]
60.
Pe ez-He nandez, D.; Gu i
é
ez-V
á
zquez, C.; Jo ge, I.; L
ó
pez-Ma
í
n, S.; U sa, A.; S
á
nchez-Mad id, F.;
V
á
zquez, J.; Y
á
ñez-M
ó
, M. The in acellula in e ac ome o e aspanin-en iched mic odomains e eals hei
unc ion as so ing machine ies owa d exosomes. J. Biol. Chem. 2013,288, 11649–11661. [C ossRe ]
61.
She chenko,A.; Wilm, M.; Vo m, O.; Mann,M.MassSpec ome icSequencing o P o eins om Sil e -S ained
Polyac ylamide Gels. Anal. Chem. 1996,68, 850–858. [C ossRe ] [PubMed]
62.
O ea, I.; Ruiz-S
á
nchez, I.; Cañe e, R.; Caballe o-Villa aso, J.; Cañe e, M.D. Iden i ica ion o candida e
se um bioma ke s o childhood-onse g ow h ho mone de iciency using SWATH-MS and ea u e selec ion.
J. P o eom. 2018,175, 105–113. [C ossRe ] [PubMed]
63.
Meye , J.G.; Schilling, B. Clinical applica ions o quan i a i e p o eomics using a ge ed and un a ge ed
da a-independen acquisi ion echniques. Expe Re . P o eom. 2017,14, 419–429. [C ossRe ] [PubMed]
64.
Luo, Y.; Mok, T.S.; Lin, X.; Zhang, W.; Cui, Y.; Guo, J.; Chen, X.; Zhang, T.; Wang, T. SWATH-based p o eomics
iden i ied ca bonic anhyd ase 2 as a po en ial diagnosis bioma ke o nasopha yngeal ca cinoma. Sci. Rep.
2017,7, 1–11. [C ossRe ] [PubMed]
65.
Ka pie i ch, Y.V.; Dabney, A.R.; Smi h, R.D. No maliza ion and missing alue impu a ion o label- ee
LC-MS analysis. BMC Bioin o m. 2012,13, S5. [C ossRe ] [PubMed]
©
2019 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 p://c ea i ecommons.o g/licenses/by/4.0/).
