Proteins Driving Stem Cell Differentiation:

Characterising the Chromatin Proteome of

Differentiating NT2 Cells
Abstract
This study aimed to characterise the chromatin proteome of retinoic acid-induced differentiating
NT2 cells over five time points, using a newly developed, optimised, label-free approach. NT2
cells are a model system for studying stem cells, providing a clearer picture of the underlying
mechanisms involved in the differentiation of pluripotent stem cells into a homogenous population
of a defined cell type neuronal cells. Stem cells, with their unique properties of pluripotency and
self-renewal, potentially represent a limitless supply of cells for regenerative therapies to treat a
range of human diseases such as neurodegeneration, heart disease and cancer. owever, the
widespread clinical application of stem cells has been persistently undermined by a lac! of full
comprehension of the underlying mechanisms responsible for the maintenance of pluripotency and
the directed differentiation of stem cells into specific cell types. "ifferentiation fundamentally
involves the protein-driven epigenetic regulation of gene e#pression, repressing pluripotency
genes while activating cell lineage-specific genes. $s such, proteomics approaches represent a
powerful tool for tac!ling these limitations, providing a quantitative systems-wide perspective.
%ur study charted changes in protein abundance during differentiation in an effort to determine
the mechanisms behind the earliest events, which lead to a loss of pluripotency and subsequently
to the acquisition of a specific cell fate. %ur approach resulted in the identification of &2'((
proteins and their differential e#pression patterns which, to our !nowledge, is the deepest
proteome coverage of differentiating NT2 cells to date. $mong the identified proteins were the
three well-established pluripotency regulators, %ct', Nanog, and So#2. )rom reviewing previous
literature it would appear that this is the first instance in which Nanog has been identified in the
proteome of differentiating NT2 cells. $ll three were found to be down-regulated as
differentiation proceeded. The ma*ority of subunits from a number of different established
chromatin remodelling comple#es were also identified, presenting the opportunity to observe
2
whether cell-type specific subunit e#changes were occurring during the differentiation process.
The e#pression and interaction profiles of a subset of proteins were e#amined, to provide an
e#ample of the information which can be derived from our large dataset. )urthermore, over +((
previously uncharacterised proteins were identified, demonstrating the efficacy of our optimised
label-free approach, and providing potentially novel putative stem cell factors for further
e#amination. The identification of novel stem cell factors and regulatory networ!s and their
mechanisms-of-action could provide new insights into the process of differentiation, potentially
ta!ing us one step closer to a better comprehension which could benefit the clinical application of
stem cells.
,
Table of Contents
0.Abstract 1
1. Introdction !
+.+ Stem cell properties -
+.2 .pigenetics and stem cell phenotype
-
+., /roteomics approaches to studying stem cells 0
2. "aterials and "ethods 10
2.+ 1ell culture and 2$-mediated cell differentiation +(
2.2 1hromatin 3solation +(
2., Trichloroacetic acid 4T1$5 precipitation ++
2.' S"S-/$6. fractionation ++
2.- 3n-gel trypsin digestion and peptide e#traction +2
2.0 7ass spectrometry 47S5 analysis +'
2.8 /rotein identification and relative quantification +-
#. $eslts 1%
,.+ /roteomics approach to quantifying the changes in chromatin proteins
during differentiation.
+0
,.2 7ascot protein identification and quantification
+8
,., The dynamics of 92'(( proteins mapped in differentiating NT2 cells
+:
,.' ;uantification of differentiating NT2 cells
+:
,.- ;uantification of interacting proteins as differentiation proceeds 2'
!. Discssion #1
'.+ The current status of stem cell proteomics ,2
'.2 $n optimised proteomics approach ,2
'., )uture applications and routes of enquiry ,,
'.' 1onclusions ,-
%. &ibliogra'h( #%
). A''endices !0
1. Introdction
'
Stem cell properties
Stem cells have been a popular research topic for several decades. 3n spite of this there are still
many unanswered questions in regards to the regulation of their unique properties<pluripotency
and self-renewal<and the mechanisms directing single-lineage 4homogenous5 differentiation.
/luripotency is the ability to transform from an undifferentiated state =+> into any type of cell in
the adult body, while self-renewal constitutes the ability to replicate into an undifferentiated state.
?ith these properties, stem cells represent a potentially limitless source of new cells with which to
treat an array of human diseases and in*uries such as heart disease =2>, neurodegeneration =,>
and holds great potential for regenerative medicine =',-> 4)igure +.+5. owever, in order to
seriously consider stem cells in the conte#t of therapeutics, a more profound understanding of the
mechanisms which regulate pluripotency, self-renewal and homogenous differentiation is
required.
*igre 1.1. Stem cells have great 'otential for se in regenerative medicine+ and for std(ing
hman develo'ment and cancers.
-
To overcome these obstacles, the molecular mechanisms which drive differentiation need to be
studied in more depth. 6enomic "N$ sequences between different cell types in an organism are
almost identical@ hence it would appear that epigenetic modifications could e#plain much of the
difference between cells that have highly varying roles in the body =0>. .pigenetic modifications
are heritable changes which do not change the "N$ sequence, but influence gene e#pression by
controlling the access of transcriptional machinery to gene promoters through alterations to the
chromatin structure. "ifferentiation of a stem cell into any defined cell type involves specific
alterations to gene e#pression, in order to repress pluripotency genes and activate cell type-
specific genes 4)igure +.25@ this is driven by epigenetic modifications ultimately brought about by
protein factors and comple#es, ma!ing proteomic-based approaches ideal for studying the process
of differentiation.
*igre 1.2. Sim'lified differentiation model.
Epigenetics and stem cell phenotype
Several types of epigenetic modifications can be responsible for altering gene e#pression@ $T/-
dependent chromatin remodelling involves chromatin remodelling comple#es which alter the
association of the core histones in nucleosomes to the "N$ wound around them, changing the
0
structure of the chromatin =8>. istone modification, whereby !ey amino acid residues on the
histone tails are altered, is another form of epigenetic modification typically involving histone
acetylation or methylation < modifications which change the interaction between the "N$ and
histones, leading to altered chromatin structure =:>.
Proteomics approaches to studying stem cells
The ma*ority of previous studies have focused on mouse 4m.S1s5 or human 4h.S1s5 embryonic
stem cells, mostly loo!ing at a small subset of genes andAor proteins, and only a few loo!ing at the
proteome of differentiating cells. The firm establishment of several !ey pluripotency-inducing
transcription factors by Bamana!a 42((0, 2((85, including %ct' and So#2, provided a basis from
which to study pluripotency and the mechanisms involved its regulation =C,+(>. Their wor!
focused on the use of a few defined !ey factors to induce a pluripotent state onto already
differentiated somatic cells@ the resulting cells coined induced pluripotent stem cells. %ther groups
such as van den Derg et al. and /ardo et al. 42(+(5 used )E$6-affinity proteomics approaches to
e#amine the interactors of the !ey pluripotency factor %ct' in .S1s, aiming to determine the
networ! of proteins which regulate FstemnessG =++,+2>. The results of these studies indicated a
large degree of interaction between numerous transcription factors, with many showing co-
occupancy of same gene promoters. %verlapping interaction of pluripotency factors with several
remodelling comple#es such as Nu2" and hS?3ASN) was also observed, suggesting the
coordinated activity of a comple# networ! of proteins in regulating and maintaining an
undifferentiated state. These studies highlighted the benefits of a proteomics perspective as a
powerful tool through which to observe the underlying mechanisms driving pluripotency.
.#pression proteomics, at its core, aims achieve three levels of protein analysesH separation,
quantification, and ultimately identification. Traditionally these approaches have largely involved
two-dimensional 42"5 separation methods such as 2"-liquid chromatography and the gel-based
8
2"-/$6. or 2"-differenitial gel electrophoresis 42"-"36.5. 7ore recently, isotope label-based
approaches such as S3E$1 and iT2$; have become popular =+,>. Several recent studies have
used these approaches to e#amine and quantify the proteome of h.S1s, to better understand how
proteins regulate differentiation =+',+->.
$lthough h.S1s are what we ultimately wish to understand, they are not always the most
straightforward modelH they differentiate into any cell type but it is not yet understood how to
effectively manipulate this in vitro. This ma!es it difficult to reproduce results, limits clinical
merit, and complicates obtained results due to the mi#ture of signals from different cell lineages.
$side from these technical issues, there are also ethical problems with the use of h.S cells in
many countries. $nother cell line, NT2 4or NT.2$-25, can be used to overcome these difficulties.
NT2 is a well-characterised, immortalised human embryonal cancer cell line. ?hen treated with
retinoic acid 42$5, NT2 cells can be induced to undergo differentiation and are hence said to be a
malignant equivalents of h.S1s 4both are capable of pluripotency and self-renewal5. $ ma*or
advantage of NT2 cells, however, is that they undergo homogenous differentiation<
differentiating only along a single lineageH the neuronal lineage 4)igure +.,5, ma!ing it a more
easily replicated and less comple# model system for stem cell differentiation =+0,+8>.
:
*igre 1.#. Com'arison of heterogenos ,S cell differentiation vs homogenos NT2 cell
differentiation.
3n this study we developed a new optimised approachH the use of a label-free, single dimensional
4+"5 gel-based system which has shown greater resolution and sensitivity in quantifying protein
e#pression. Ising this approach we were able to quantitatively assay the nuclear proteome of
differentiating NT2 cells and identified &2,'(( proteins, and their e#pression profiles, over five
stages of differentiation 4day (, +, 2, ', and :5. %ur catalogue of proteins included established
pluripotency factors such as %ct', Nanog, So#2 and Sall' =C-+2,+C>. The ma*ority of subunits
from remodelling comple#es lin!ed to the regulation of pluripotency and self-renewal were also
identified, including those of Nu2", Sin,, hS?3ASN), and D2$)-"$1 =2+-2->. These results
have shown the efficacy of our label-free approach in providing both high resolution and deep
coverage of the nuclear proteome, in a model system which is efficient and readily reproducible.
C
.#amination of the e#pression profile of a subset of &-( proteins provided an e#ample of the
insight which can be derived from our e#tensive dataset. ere we monitored the e#pression and
interactions of a number of transcription factors and subunits of chromatin remodelling
comple#es, to generate a temporal interaction networ! which illustrates a high degree of
prospective cooperation between these proteins in their regulation of pluripotency.
+(
2. "aterials and "ethods:
$ll materials were purchased from Sigma-$ldrich.
Cell culture and RA-mediated cell differentiation
NT2 cells were cultured on +'cm
2
plates using "7.7 with +(J )DS and +J
/enicillinAStreptomycin. omogenous differentiation into the neuronal lineage was induced by
seeding asynchronously growing cells at ,(J confluency, treating with +(K7 all-trans retinoic
acid 42$5 dissolved in "7S% after 2' h. 1ells were incubated with 2$ for (, +, 2, ', and : days
and harvested at C(-+((J confluency.
Chromatin isolation
Table 2.1. Lysis Buffer compositions
Buffer A Buffer S1 Buffer S2 Buffer D Salt
10 mM Hepes
pH7.9
20 mM Hepes pH
7.9
20 mM Hepes pH 7.9 20 mM Hepes
1.5 mM MgCl2 120 mM NaCl 420 mM NaCl 1.5 mM MgCl
2
10 mM KCl 1.5 mM MgCl2 1.5 mM MgCl2 0.2 mM EDTA
0.5 mM DTT 0.2 mM EDTA 0.2 mM EDTA 10% Glycerol
10 % Glycerol!
a"" a#$er
#rac$%o&a$%o&'
10 % Glycerol 10 % Glycerol
-ml /DS was pipette on each plate prior to harvesting. arvested cells were centrifuged at 2,(((
rpm for - min at '(1 and the supernatant removed. $nother /DS wash was performed before
centrifugation at 2,-(( rpm at 'L1 for -min 4all subsequent centrifugation carried out this way5.
The e#traction of the chromatin-bound fraction involved a three-step process.
a- C(to'lasmic e.traction:
The pac!ed cell volume 4/1M5 was estimated and the pellet resuspended in a volume of Duffer $
2-, times the /1M. 1ells were incubated for +- min on ice, lysed with : stro!es of "ounce
++
homogeniser 4type $ pestle5 to brea! the cell membranes, then centrifuged@ the resulting
supernatant contained the cystoplasmic proteins 4!ept and froNen at -:(L1 in +(J glycerol5 while
the pellet contained the nuclear fraction.
b- Nclear solble fraction e.traction:
The pellet was washed with Duffer $ 4+( # /1M5 and centrifuged once as normal, then at ',-((
rpm after removing supernatant. Duffer S+ 42-, # /1M5 re-suspended the pellet and the suspension
was lysed with +- stro!es of a "ounce homogeniser 4type D pestle5 to brea! up the nucleus,
followed by centrifuged for +- min at ',-(( rpm@ the supernatant contained the nuclear soluble
proteins 4!ept and froNen5.
c- Chromatin/bond 'rotein e.traction:
The pellet was centrifuged for 2 min at ',-(( rpm, to remove any residual contamination, then re-
suspended in S2 buffer 42-, # /1M5 and incubated for ,( min on a wheel, at 'L1. The lysate was
diluted to 22(m7 Na1l by the addition of Duffer ". DenNonase Nuclease 42-( IAKl5 was added to
a final concentration of (.+ IAKl, followed by incubation for + h 2- min at 'L1. The insoluble
fraction was separated from the e#tract by centrifugation at ',-(( rpm for +- min. The
supernatant, containing the soluble faction, was stored at -:(L1 in +(J glycerol.
Trichloroacetic acid (TCA) precipitation
T1$ protein precipitation concentrated the NT2 chromatin proteins prior to S"S-/$6.. 2(J
T1$ solution was prepared from stoc! powder 42.(g in +( ml of d
2
%5. 2(( Kl of each chromatin
sample was pipetted into separate (.-ml labelled 4(d, +d, 2d, 'd, :d5 tubes. 2(( Kl 2(J T1$ was
added to each, followed by ,( min incubation on ice then centrifugation at +0,((( rpm, 'L1 for ,(
min. $ll subsequent centrifugation was carried out this way. 2-( Kl cold acetone wash was done
on each pellet to remove contaminants.
SDS-PAGE fractionation
+2
2(( Kl 2# concentrate Eaemmli sample buffer and +( Kl 2-7ercaptoethanol 4-J5 was pipetted
into a (.- ml tube and mi#ed. T1$ precipitated samples were removed from ice. Neutral p was
essential for successful S"S-/$6. fractionation@ a p-sensitive buffer indicated neutral 4blue5 or
acidic 4green-yellow5 p. 2( Kl of sample buffer solution was added to the pellet of the one
sample and vorte#ted. $ slight colour change towards green occurred, so +ul of +7 Tris base
4Sigma5 was added to ensure a neutral p. Subsequently, 2( Kl buffer solution and + Kl of Tris
base were added to all other samples which were boiled on C-L1 boiling plate for - min, to
energise the molecules.
Table 2.2. 10X SDS-PAG-!PS "unnin# Buffer composition
$omponent %olecular &ei#'t Amount a((e( &ater nee(e(
Tr%s )ase 121 g*mol 121.0 g )ro+g,$ +p $o 1 - .y
HE/E0 212 g*mol 210.0 g a""%$%o& o# +l$rap+re
0D0 222 g*mol 10.0 g 3a$er
Table 2.). 1X SDS-PAG-!PS "unnin# Buffer
composition
$omponent $oncentration *+erall p!
Tr%s )ase 100 mM pH 2.0
HE/E0 100 mM
0D0 41 mM 0.1%'
+O S"S-/$6.-./.S running buffer was preparedH first +(O S"S-/$6.-./.S running
buffer was made 4Table 2.25. $ +(-fold dilution, with ultrapure water, resulted in the +O running
buffer 4Table 2.,5. The S"S-/$6. was assembled with a '-2(J 4Diorad5 gel, and the running
buffered added. +( Kl of a broad range Diolabs standard protein mar!er was loaded into lane +. 2(
Kl of each sample, stained with 6el1ode Dlue was loaded starting from lane ,. The apparatus ran
at +(( M until the dye front had travelled P down the gel. The gel was rinsed before storing in
d
2
%, and placed on a sha!er overnight.
n-gel trypsin digestion
+,
Table 2.,. -!
,
!$*
)
Buffer solution concentrations
Buffer concentration. 200 m% 100 m% /0 m% 20 m%
0olume of ultrapure
1ater
40 ml 50 ml 40 ml 20 ml
Amount of -!
,
!$*
)
0.5124 g 0.195 g 0.152 g 0.0115 g
The wor! area was frequently cleaned with freshly prepared 8(J .t% 4ethanol5 to ensure
quality results. $ll buffers were prepared freshly 4Table 2.'5. .ach of the - lanes of gel were cut
up into +0 pieces. %ne lane was prepared at a time, using the following protocol. The day ( 4(d5
laneH +(( Kl 2(( m7 N
'
1%
,
added to each of the +0 microfuge tubes 4labelledH (d+, up to
(d+05. (d was e#cised and cut up into +mm
2
pieces 4gives large surface area for trypsin to act on5,
as outlined in )igure 2.+ followed by trypsin digestion.
*igre 2.1. Schematic of lane ctting0 lane 0d e.am'le.
Samples were prepared for in-gel trypsin digestion as followsH the total :( eppendorfs, containing
gel pieces and 2(( m7 N
'
1%
,
were placed on a ,8L1 8(( rpm thermomi#er for +(min 4all
subsequent thermomi#ing carried out this way5. The dye-containing supernatants were discarded
before adding +(( Kl 2(( m7 N
'
1%
,
A7e1N 42H, vAv5 and thermomi#ing to immobilise the
proteins. Supernatants were discarded 4done after each reagent added and
thermomi#ingAincubation5 then +(( Kl -( m7 N
'
1%
,
was added followed by thermomi#ing.
+'
+(( Kl 7e1N, a polar solvent, was then added to further dehydrate for 2 min. -( Kl +( m7 "TT
4(.(+-'g in +( ml d
2
%5 was added as a reducing agent to prevent the formation of cysteine
bonds, and incubated for 0( min at 0(L1. -(Kl -( m7 3$$A+(( m7 N
'
1%
,
4(.('0 g 3$$ in -
ml N
'
1%
,
5 was added and incubated in the dar! at room temperature for ,( min to stabilise
the reduced state. Samples were washed with ,( Kl +(( m7 N
'
1%
,
in a thermomi#er for
+(min, followed by ,( Kl 2( m7 N
'
1%
,
A7e1N 4-(H-( vAv5. Ne#t, a final dehydration 4+(( Kl
7e1N for - min5 to aid the diffusion of the trypsin into the gel. )inally, -( Kl of prepared trypsinA
N
'
1%
,
solution was added to each sample followed by overnight thermomi#ing. 2( Kg of
stoc! trypsin 4/romega5 in +(( Kl reconstitution buffer was used for preparation, aliquoted into
ten +( Kl tubes. 2-( Kl -( m7 N
'
1%
,
was added to four aliquots to generate the final
solutions.
Peptides e!traction for "S#
"igested samples were centrifuged for +( s and the peptide-containing supernatant transferred into
(.- ml tubes. 6el pieces were retained and ,( Kl 8(J 7e1N with 'J formic added to each,
waiting +( min before centrifuging for +( s to e#tract residual peptides. Supernatants from the gel-
containing tubes were combined with their other respective supernatants. +0 tubes containing
supernatants were dried down in a vacuum centrifuge at 0(L1 for 2 h. The resulting peptide pellets
were re-suspended in 2( Kl (.+J formic acid and transferred into 7S vials. The samples were
!ept at -'L1 and ready for 7S analysis.
"ass spectrometry ("S) analysis
$n .$SBnE1 with a nano-electrospray ionisation source 4/ro#eon Diosystems5 and an ET;
%rbitrap 1lassic mass spectrometer 4Thermo Scientific5 was used to perform liquid
chromatography-tandem mass spectrometry 4E1-7SA7S5. /eptides were separated on a +-cm
reversed phase analytical column 48- Km internal diameter5 in-house pac!ed with , Km 2eproSil-
+-
/ur 1+:$; beads, employing a + h gradient from 2-2-J buffer D 4CC.CJ 7e1NA(.+J formic
acid5 at a flow rate of (.' KlAmin.
"ata was continuously collected in a data-dependent manner, with the %rbitrap collecting a survey
scan at 0(,((( resolution with an automatic gain control 4$615 target of + # +(
0
. 1ollision
induced dissociation 413"5 7A7S scans followed, using the +( most abundant ions from the
survey scan, with an $61 target of -,(((@ signal threshold of +,(((@ 2.( "a isolation width@ and
7S activation time at ,-J normalised collision energy. 1harge state screening was used to re*ect
unassigned or +Q charge states. "ynamic e#clusion was enabled to ignore masses for ,( s that had
previously selected for fragmentation.
Protein identification and relati$e %uantification
2aw 7S files were processed into 7ascot 6eneric )ormat 4.mgf5 files by 7ascot "istiller 47atri#
Science5, and used to query 7ascotRs 7SA7S 3on Search 4v2.,.(25. /roteins were identified by
comparing observed peptides to the Ini/rot protein database 4release -8.+-@ -,',2'2 sequences5.
"efault parameters were used, with carbamidomethylation set as a fi#ed modification and
o#idation 475 and $cetyl 4/rotein N-term5 as variable modifications. /eptide mass tolerance was
,( ppm, fragment mass tolerance 47SA7SA tol.5 (.- "a and two missed cleavages were allowed.
"ynamic e#clusion was used to e#clude detected ions from repeat fragmentation for 2 min. The
principle of ma#imum parsimony ='C> was used in matching peptides to proteins in the database,
with proteins identified in at least two of three e#perimental datasets being accepted.
3dentifications which were only based on one unique peptide, or two 4or more5 unique peptides in
only one dataset, were manually validated by gauging the assignment of ma*or pea!s, the
occurrence of continuous 4at least three amino acids5 y- or b-ion series, low mass error 4S-((
ppm5, and the p-value. /roteins were deemed significant if the p-value was lower than the
Donferroni ad*usted threshold of (.(-An 4where n is the number of tests performed5.
+0
&' Results
Proteomics approach to %uantifying the changes in chromatin proteins during differentiation
*ig. #. 1or2flo3 for 4antitativel(
characterising the NT2 chromatin 'roteome.
5 image from Serra et. al 62007- 8!9:
; images from Conce'ts of <enetics+ 9th edn
6200=- 8!7:
> image from The &iotechnolog( Pro?ect at
"ATC 6htt':@@biotech.matcmadison.ed-
+8
"ascot protein identification and %uantification analysis
.#amination of any proteins from the 7ascot identification, %ct' 4/%-)+TI7$N5 for
e#ample, gave more information about its identification 4)igure ,.+5, allowing for the accuracy of
the identification to be ascertained. 7ascot assigned each potential protein identification a
F7ascot ScoreG, based on several factors includingH number of peptides matched to the protein
sequence, mass error, signal-to-noise ratio, mass pea! assignment, and ion assignment. These
traits could also be individually e#amined to ensure accuracy. %ct' provided a good e#ample of
accurate protein identificationH it had a high 7ascot score of +-C 4where, typically in proteomics
literature, proteins with a score above ,( are considered to be high quality matches5 and 0
different peptides matching to the %ct' protein sequence. )urther, e#amination of the 0 matched
peptides showed the high quality of these 4)igure ,.+5.
*igre #.1. The Protein Aie3 6to' left-+ sho3ing the "ascot Score 6highlighted-. The Pe'tide
Aie3 6to' right- for a s'ecific matched 'e'tide+ sho3ing the assignment of most 'ea2s
+:
6highlighted-. Continos (/ and b/ion series 6highlighted- and the lo3 6B=00 ''m- mass
errors 6bottom-.
The dynamics of ()*++ proteins mapped in differentiating ,T) cells
$ total of 2''0 proteins were identified in the chromatin proteome of differentiating NT2 cells,
using E1- 7SA7S. )or each differentiation time point 4day (, +, 2, ', :5, peptides were identified
by reference to a human amino acid sequence database and mapped to these 2''0 proteins as
shown in Table ,.+. $n e#cerpt from the complete catalogue of proteins is available in $ppendi#
+.
Table ).1. Total proteins an( pepti(es i(entifie( by %S
analysis
Days of
Differentiation
Day
0 Day 1 Day 2 Day , Day 2
Total Proteins 2445 2445 2445 2445 2445
Total Pepti(es
1011
7 9915 11117 9922 9047
Table #.1. Total 'roteins and 'e'tides in the chromatin 'roteome of differentiating NT2
cells+ for each time 'oint.
-uantification of differentiating ,T) cells#
$ subset of the identified proteins was e#amined in order to verify our results. This included
!nown pluripotency factors, previously described proteins associated with neuronal cell-specific
development, and !nown chromatin remodelling comple#es =:-+2,+:-,C>.
To verify that differentiation along the neuronal lineage had occurred, the relative peptide levels
of pluripotency factors and neuronal-specific proteins =,+-,C> were observed 4Table ,.25. $s
e#pected from pluripotent NT2 cells induced to differentiate into neuronal-lineage cells, proteins
!nown to be associated with pluripotency decreased in abundance as differentiation proceeded,
while proteins associated with neuronal cell development increased in abundance 4)igures ,.2 and
,.,5.
+C
Table ).2. Pluripotency-associate( transcription factors an( -euronal-associate(
proteins
Protein Primary 3unction
*+erall
4pression
$'an#e
"eferenc
e5s6
Transcription
3actors
6c$4
Key pl+r%po$e&cy $ra&scr%p$%o& #ac$or
"o3&
27
12!12!25
Na&og
Key pl+r%po$e&cy $ra&scr%p$%o& #ac$or
"o3&
27
12!12!25
0o82
Key pl+r%po$e&cy $ra&scr%p$%o& #ac$or
"o3& 2712!25
0all4
Tra&scr%p$%o& reg+la$%o&9 %&$erac$s 3%$,
Na&og
"o3&
11!12!19!2
5
0all1
Tra&scr%p$%o& reg+la$%o&9 $,o+g,$ $o %&$erac$
3%$, compo&e&$s o# $,e N+:D c,roma$%&
remo"ell%&g comple8
"o3& 19!20
Ar%"1.
Tra&scr%p$%o& reg+la$%o&9 %&$erac$s 3%$, 6c$4
"o3& 11711
Dp#2
Tra&scr%p$%o& reg+la$%o&9 %&$erac$s 3%$, 6c$4
"o3& 11711
:%#1
Tra&scr%p$%o& reg+la$%o&9 %&$erac$s 3%$, 6c$4
"o3& 11711
-euronal-
associate(
Proteins


Nes$%&
:e;+%re" #or .ra%& a&" eye "e<elopme&$9
Ne+roge&es%s
+p 11
Gap41
Assoc%a$e" 3%$, &er<e gro3$,9 Ner<o+s
sys$em "e<elopme&$
+p 11
A,&a=
Ne+ro&al cell "%##ere&$%a$%o&9 Ner<o+s
sys$em "e<elopme&$
+p 12
G#ra1
A8o& g+%"a&ce
+p 15!15
Kl=5
Ce&$ral &er<o+s sys$em "e<elopme&$9
Myel%&a$%&9 :eg+la$%o& o# cell "%##ere&$%a$%o&
+p 17
Ac$&1
)ra%&7spec%#%c %so#orm9 co&$a%&s .o$, smoo$,
m+scle a&" &o&7m+scle e8o&s $o g%<e a
.ra%&7spec%#%c se;+e&ce "oma%&.
Cy$os=ele$al pro$e%&.
+p 14
:$&1
May .e %&<ol<e" %& cere.ell+m
"e<elopme&$9 May co&$r%.+$e $o &e+ro&al
s+r<%<al a&" plas$%c%$y
+p 12
Cra.p2
:eg+la$es $,e access o# re$%&o%c ac%" $o $,e
&+clear re$%&o%c ac%" recep$ors
+p 11
No<a2
Ne+ro&7spec%#%c :NA .%&"%&g pro$e%&9 May
reg+la$e &e+ro&al m%gra$%o&
+p 19
Table #.2. Characterising 'lri'otenc( transcri'tion factors and neronal/associated
'roteins in differentiating NT2 cells.
2(
*igre #.2. Do3nreglation of 'lri'otenc(/associated transcri'tion factors as NT2
differentiation 'roceeds.
*igre #.#. C'reglation of 'roteins associated 3ith the develo'ment and fnction of
neronal lineage cells+ as NT2 differentiation 'roceeds.
To further verify results, the relative protein levels of %ct' and Nanog were compared to the
m2N$ levels which we e#amined in a previous study using q/12 during neuronal differentiation
of NT2 cells 4)igure ,.'5. %ur %ct' e#pression data correlated with this pattern, showing
progressive downregulation and a near-diminished e#pression by day '. Nanog showed a very
2+
similar pattern to the m2N$ data, with down-regulation by day 2, however the levels proceeded
to be slightly upregulated again in day :. This is upregulation is li!ely artefactual, however, being
based on only a single peptide detection event.


*ig. #.!. Com'arison of relative Dct! and Nanog m$NA and 'rotein levels as NT2
differentiation 'roceeds.
.igh resolution and co$erage of the chromatin proteome of differentiating ,T) cells#
$ primary aim of this study was to develop an optimised approach for quantitatively
characterising the proteome of differentiating cells. %ur dataset was e#amined for the presence of
normally low abundance proteins such as the !ey pluripotency factors %ct', So#2 and Nanog. $ll
22
three proteins were present in our dataset 4Table ,.,5, indicating that a superior resolution had
been achieved.
Table ).). 7ey pluripotency transcription factors i(entifie(
Protein Day 0 Day 1 Day 2 Day , Day 2
Transcription 3actors
6c$4 5 5 2 2 0
Na&og 2 2 0 0 1
0o82 4 2 5 4 0
Table #.#. All three lo3 abndance transcri'tion factors 3ere sccessfll( identified in the
NT2 chromatin 'roteome+ over five 'oints of differentiation.
The level of coverage achieved in our dataset was notably high, as illustrated by the 2,''0 proteins
identified. The dataset included both low- and high-abundance proteins, and the ma*ority of core
and associated subunits from a number of !nown chromatin remodelling comple#es 4Table ,.'5.
$lso included were over +(( previously uncharacterised proteins, further supporting the high
coverage of the dataset.
Table ).,. "emo(ellin# comple4es i(entifie( by mass spectrometric analysis of
(ifferentiatin# -T2 cells
Protein Primary 3unction *+erall
4pressio
n $'an#e
"eferenc
e5s6
'S&89S-3
$omple4



0marca2
Tra&scr%p$%o&al Coac$%<a$or
"o3& 42
0marca4 )rg1'
Tra&scr%p$%o&al Coac$%<a$or
"o3& 12!25!42
0marc"1
Tra&scr%p$%o&al ac$%<a$%o&*repress%o& .y
c,roma$%& remo"ell%&g
"o3& 42
Ar%"1a Tra&scr%p$%o&al ac$%<a$%o&*repress%o& .y
c,roma$%& remo"ell%&g9 No&7spec%#%c DNA
.%&"%&g
"o3& 25
0marcc1 Tra&scr%p$%o&al ac$%<a$%o&*repress%o& .y
c,roma$%& remo"ell%&g9 May s$%m+la$e
AT/ase ac$%<%$y
"o3& 12!42
0marcc2 Tra&scr%p$%o&al ac$%<a$%o&*repress%o& .y
c,roma$%& remo"ell%&g9 May s$%m+la$e
AT/ase ac$%<%$y
"o3& 42
2,
0marc.1
Core compo&e&$ o# 0>?*0N@ comple89
C,roma$%& remo"ell%&g
"o3& 25!42
0marce1
Tra&scr%p$%o&al ac$%<a$%o&*repress%o& .y
c,roma$%& remo"ell%&g
"o3& 42
0marca5
AT/ase ac$%<%$y %& AT/7"epe&"e&$
&+cleosome7remo"ell%&g
"o3& 11
Braf)/-!DA$
5B!$6 $omple4


)ra#15 Hgm20.'
:e;+%re" #or :coc1 me"%a$e" repress%o& o#
&e+ro&al spec%#%c ge&e promo$ers9 DNA7 a&"
pro$e%&7.%&"%&g
"o3& 25!44
Table ).,.
$ontinue(
),c20 /,#21a'
C,roma$%& remo"ell%&g9 Tra&scr%p$%o&
reg+la$%o&9 May ac$ as a sca##ol" %& )HC
"o3& 41!44
-s"1 K"m1a'
H%s$o&e "eme$,ylase! "eme$,yla$es H1K4me
a&" H1K9me9 Coac$%<a$or*Corepressor
"o3& 25!44
:cor1 Co:E0T'
Esse&$%al compo&e&$ o# )HC comple89
Tra&scr%p$%o&al repress%o&
"o3& 25!41!44
H"ac1
H%s$o&e "eace$ylase
&o&e 25!41!44
H"ac2
H%s$o&e "eace$ylase
&o&e 25!41!44
Sin)-!DA$
$omple4

0%&1a
Tra&scr%p$%o&al repressor9 ?&$erac$s 3%$, Ma"7
Ma8 ,e$ero"%mer $o $e$,er %$ $o DNA9
Corepressor #or :es$
"o3& 21!44
H"ac1
H%s$o&e "eace$ylase
&o&e 21!44
H"ac2
H%s$o&e "eace$ylase
&o&e 21!44
0ap10
:ecr+%$me&$ o# 0%&17HDAC comple8 $o a
spec%#%c s+.se$ o# Ncor corepressor
comple8es
&o&e 21!44
0ap12
E&,a&ces $,e a.%l%$y o# 0%&17HDAC7me"%a$e"
$ra&scr%p$%o&al repress%o&9 Ca& "%rec$ $,e
#orma$%o& o# $,e comple8 $o core ,%s$o&es
&o&e 21
0ap110
Tra&scr%p$%o&al repressor9 May #+&c$%o& %& $,e
assem.ly a&"*or e&Ayma$%c ac$%<%$y o# $,e
comple8! or %& me"%a$%&g %&$erac$%o&s
.e$3ee& $,e %$ a&" o$,er comple8es
"o3& 21!44
0+"s1
Tra&scr%p$%o&al repressor9 A+gme&$s H"ac1
ac$%<%$y9 May #+&c$%o& %& $,e assem.ly a&"*or
e&Ayma$%c ac$%<%$y o# $,e comple8! or %&
me"%a$%&g %&$erac$%o&s .e$3ee& $,e %$ a&"
o$,er comple8es
&o&e 21!44
Ar%"4.
Tra&scr%p$%o&al repressor9 May #+&c$%o& %& $,e
assem.ly a&"*or e&Ayma$%c ac$%<%$y o# $,e
comple8! or %& me"%a$%&g %&$erac$%o&s
.e$3ee& $,e %$ a&" o$,er comple8es
&o&e 21!44
:..p4
)%&"s ,el%8 1 o# ,%s$o&e H49 May $arge$ o$,er
N+:D s+.+&%$s $o ,%s$o&es
&o&e 21
:..p7
)%&"s ,el%8 1 o# ,%s$o&e H49 May $arge$ o$,er
N+:D s+.+&%$s $o ,%s$o&es
&o&e 21!44
?&g1
?&<ol<e" %& a &ega$%<e reg+la$ory pa$,3ay o#
cell gro3$,! 3%$, p51
"o3& 44
?&g2
?&<ol<e" %& p51 ac$%<a$%o& a&" p517
"epe&e"e&$ apop$o$%c pa$,3ays
"o3& 44
2'
)rms1
Tra&scr%p$%o&al repressor9 /romo$es H"ac1
.%&"%&g $o promo$er reg%o&s
"o3& 44
)rms1l
?&<ol<e" %& H"ac17"epe&"e&$ $ra&scr%p$%o&al
repress%o&
"o3& 44
Table #.!. Sbnits of chromatin remodelling com'le.es0 chromatin remodelling is a 2e(
'art of controlling gene e.'ression+ b( reglating the access of transcri'tional machiner( to
gene 'romoters.
-uantification of interacting proteins as differentiation proceeds
The dataset produced by our optimised approach provides a strong basis for further studies to
elucidate the underlying mechanisms of pluripotency and directed differentiation of stem cells.
Incharacterised proteins identified in the dataset can be e#amined for their role in these processes
and the quantitative protein data can be used in a number of ways, including studying the
relationship between different regulatory proteins as differentiation proceeds. To illustrate this, a
subset of -' proteins were selected and e#amined in greater detail. These proteins included !nown
pluripotency factors, neuronal proteins, and chromatin remodelling comple#es =:-+2,+:-'+>. The
interactions between these proteins were characterised in silco using a bioinformatics tool
designed for analysis of protein and gene networ!s 4ST23N6@ httpHAAstring-db.orgA5. ST23N6 uses
both !nown and predicted protein-protein interactions to generate a networ! for the user-input
proteins, with the measured interactions including both physical and functional ones. The
information for the interactions is derived from various sources such as genomic conte#ts, high-
throughput e#periments, conserved coe#pression, and from te#t-mining literature databases such
as /ub7ed. The collective interaction data is then quantitatively integrated to construct the protein
networ!.
3nteraction networ!s were created using both the confidence 4)igure ,.-5 and evidence 4)igure
,.05 analyses. 1onfidence Miew reflects the number of different e#perimental reports supporting a
connection between the proteins, while .vidence Miew reflects the number of different types of
e#perimental evidence for the interactions. 3nteraction was observed between the transcription
2-
factors and the remodelling comple#es, and clustering was observed between more closely related
proteins. The subunits of the chromatin remodelling comple#es showed clustering with one
another, with sub-clustering observed in regards to the histone deacetylating 4Nu2", Draf-"$1,
Sin,-"$15 and non-deacetylating 4hS?3ASN)5 comple#esR subunits.
20
28
The networ! generated by ST23N6 showed preliminary evidence of interaction between the
proteins associated with regulation pluripotency andAor self-renewal. Strong interaction was
observed between the three !ey transcription factors %ct' 4/%I-)+5, Nanog and So#2. Sall' was
seen to interact, with a high confidence, with both %ct' and Nanog =+C>. Sall+ appeared to act as a
possible bridge between Nanog and "$1+ and 2 =+C,2(>. Strong interaction was also present
between subunits of the same comple#, and between subunits of different comple#es =2-2,,,(>. %f
the three !ey transcription factors, %ct' and Nanog showed the most prolific interaction with
subunits of different remodelling comple#es, in congruence with previous studies =++,+2,+:,2C,,(>
4Table ,.-5. %nly 8 of the ,8 comple#esR subunits showed interaction with the transcription
factors studied. These results suggest that only a small number of the total subunits from the
various remodelling comple#es are the focus of interaction<and possible regulation<by the
transcription factors. This could, alternatively, indicate that transcription factors other than those
we studied are responsible for interacting with the other subunits of these comple#es.
Table )./. 8nteraction of t'e transcription factors 1it' subunits of t'e four
remo(ellin# comple4es
8nteractin#
Subunit
$omple4
8nteractin#
Subunit
$omple4
-ano# *ct,
H"ac2
N+:D! 0%&7HDAC! )ra#7
HDAC M."1 N+:D
M."1 N+:D K"m1a )ra#7HDAC
0%&1a 0%&7HDAC 0marca4 ,0>?*0N@
K"m1a )ra#7HDAC K"m1a )ra#7HDAC
0marca4 ,0>?*0N@ 0marca4 ,0>?*0N@
0marc"1 ,0>?*0N@
So42 Sall1
H"ac2
N+:D! 0%&7HDAC! )ra#7
HDAC H"ac1
N+:D! 0%&7HDAC! )ra#7
HDAC
0%&1a 0%&7HDAC H"ac2
N+:D! 0%&7HDAC! )ra#7
HDAC
0marca4 ,0>?*0N@
Table #.=. Interactions of the transcri'tion factors 3ith a small nmber of com'le. sbnits.
2:
The confidence analysis was used for further e#amination, being more visually comprehensible.
;uantitative protein e#pression data 4Table ,.05 was combined with the ST23N6 interaction
networ!, for each time point, to generate a temporal protein interaction networ! 4)igure ,.85. $
ST23N6 clustering algorithm, 71E U ' ='0>, was used to reduce some visual noise by causing
the -' proteins to be clustered within four distinct groupsH the transcription factors, the "$1-
activity comple#es, the non-"$1-activity comple#es, and the neuronal-related proteins
4$ppendi# 25.
Table ).:. Protein e4pression o+er fi+e time points as
;uantifie( by mass spectrometric analysis
Protei
n
Day
0
Day
1
Day
2
Day
,
Day
2 Protein
Day
0
Day
1
Day
2
Day
,
Day
2
Transcription
3actors 'S&89S-3 $omple4
6c$4 5 5 2 2 0 )ra#15 7 5 5 1 1
Na&og 2 2 0 0 1
0marca
4 12 2 9 2 1
0o82 4 2 5 4 0
0marca
2 7 1 4 4 1
0all4 27 27 27 22 1
0marc"
1 25 21 21 22 10
0all1 5 12 9 5 1
0marc"
2 14 5 11 14 4
-euronal Proteins Ar%"1a 29 21 11 20 9
Nes$%& 11 41 45 12 44
0marcc
1 42 41 42 15 29
Gap41 0 0 1 1 2
0marcc
2 25 25 21 12 14
A,&a= 59 120 112 114 159
0marc.
1 5 5 1 5 1
G#ra1 0 1 1 4 4
0marce
1 19 21 20 19 15
Kl=5 0 0 0 1 1
0marca
5 7 0 1 7 0
Ac$&1 14 22 11 29 45 Sin)-!DA$ $omple4
:$&1 0 0 1 1 1 0%&1a 5 4 1 1 2
Cra.p
2 0 0 1 1 2 H"ac1 4 1 1 1 4
No<a2 1 1 1 1 1 H"ac2 4 5 4 5 4
-u"D
$omple4 0ap10 4 1 5 5 1
C,"4 10 12 15 7 1 0ap12 1 1 4 1 1
H"ac1 4 1 1 1 4 0ap110 11 1 2 7 0
H"ac2 4 5 4 5 4 0+"s1 0 1 0 0 0
M."1 10 5 9 7 1 Ar%"4. 1 0 1 1 0
M$a1 2 1 1 1 1 :..p4 7 7 5 2 7
M$a2 1 0 2 1 1 :..p7 4 1 4 1 4
2C
Ga$a"
2a 12 2 11 2 1 ?&g1 1 2 1 1 0
Ga$a"
2. 9 5 9 2 1 ?&g2 2 1 2 1 0
:..p4 7 7 5 2 7 )rms1 1 1 2 1 0
:..p7 4 1 4 1 4 )rms1l 1 0 0 0 0
Table ).:.
$ontinue(
P"$2
$omple4 Braf-!DA$ $omple4
EA,2 1 1 1 1 0 )ra#15 7 5 5 1 1
0+A12 1 1 0 1 1 /,#21a 4 2 2 1 0
M$#2 0 1 0 0 0 K"m1a 2 1 1 1 1
0e$ 5 5 5 5 5 :cor1 14 11 11 12 2
:..p4 7 7 5 2 7 H"ac1 4 1 1 1 4
:..p7 4 1 4 1 4 H"ac2 4 5 4 5 4
Table #.%. Protein e.'ression+ as 4antified b( S'ectral Cont 6the nmber of tandem "S
events identif(ing that 'rotein- over five time 'oints.
The temporal interaction networ! generated illustrated the stage of differentiation at which each
protein was either up- or downregulated, if either. The ma*ority of proteins e#amined showed a
significant change in abundance as differentiation proceeded, but there variation in the onset of
this. Studying this temporal element of stem cell interaction networ!s can provide insights into the
mechanisms at play. )or instance, some subunits of the chromatin remodelling comple#es
appeared to be downregulated before the pluripotency-associated transcription factors. /revious
studies have shown that remodelling comple#es, such as the hS?3ASN) class D$) comple#, can
change their subunit compositions as differentiation proceeds in a stage-specific manner, allowing
them to interact with different transcription factors to facilitate differentiation into specific cell
lineages ='8>. 3n following this, it could be that a similar process was occurring here, with changes
in the subunit composition of Nu2", Sin,, Draf-"$1 and other hS?3ASN) comple#es
preceding the downregulation of the pluripotency factors and the upregulation of neuronal
proteins.
,(
,+
,2
!. Discssion
The current status of stem cell proteomics
3n past years numerous proteomics approaches have been applied to studying m.S1s and h.S1s
and to a lesser degree, NT2 cells. The most popular has often been the use of isotope label-based
approaches, such as iT2$; or S3E$1 =++-+0>. Three recent studies, by 1haer!ady et al. 42((C5,
Sar!ar et al. 42(++5 and /ewsey et al. 42(+(5 utilised different label-based approaches to study the
proteomes of stem cells.
1haer!adyRs group used iT2$;-labelling of whole cell lysates to e#amine the proteome of h.S1s
over eight stages of differentiation =+'>. They quantitatively identified +,2-+ proteins using
reverse-phase E1-7SA7S with a quadrupole time-of-flight 4T%)5 system. owever, they were
unable to identify any of the pluripotency factors 4%ct', Nanog, So#25 which are only present in
low abundances in the nucleus. The low resolution of their results was li!ely due to a lac! of pre-
analysis subcellular fractionation, combined with a label-based approach which inherently
requires the mi#ing of peptides from samples at different stages of differentiation. This severely
dilutes the peptides of low abundance proteins, especially those a5 only present in the nucleus, and
b5 present only at the earliest stages of differentiation, as is the case with the pluripotency factors.
"ilution to this e#tent would ma!e detection of these peptides e#ceedingly unli!ely.
Sar!ar et al. more recently attempted to identify the proteomes of three subcellular fractions of a
single sample of undifferentiated h.S1sH the nuclear, cytosolic, and membrane fractions =+->.
.ach fraction was separately S3E$1-labelled and combined with unlabelled protein samples from
differentiating h.S1 cells, prior to analysis with an ET;-%rbitrap OE E1-7SA7S system. Ising
this approach they were able to identify :C, nuclear proteins, +,,C8 cytosolic proteins, and +,+:-
membrane proteins, with an overall of &8(J of the total identified proteins being unique to one
fraction. %verall they achieved a better coverage 4total of 9,,((( proteins identified5 than
,,
1haer!adyRs group, li!ely due to their use of subcellular fractionation, but the coverage obtained
for each individual fraction was not much improved.
.#amination of the nuclear fraction of 2$-induced differentiating NT2 cells, by /ewseyRs group,
resulted in a relatively high resolution identification of proteins =+0>. They studied samples from
four different stages of differentiation, focusing on the first si# days. Doth iT2$; and .#acTag
labelling approaches were used, with the samples pooled for analysis by E1-7SA7S with a 1T
quadrupole-ion trap system. The coverage achieved was low, with only -' proteins identified, but
these included low abundance proteins such as %ct' and So#2 4but not Nanog5, indicating that the
obtained resolution was relatively high. Through the use of clustering and interaction analyses
they also highlighted the presence of an intricate protein-protein interaction networ! in the
regulation differentiation@ the regulation li!ely occurring by the coordinated effort of numerous
proteins, leading to the activation andAor silencing of specific target genes at defined stages of
differentiation. This provided an e#ample of one of the possible applications of proteome
databases.
The techniques used in our study were optimised to provide both deep coverage of the proteome
and high-resolution, with an emphasis on discovering novel factors, to enable to identification of
even low abundance proteins which have often been missed in other studies, including those
mentioned above.
An optimised proteomics approach
3n contrast to the studies described, we used +" gel-based protein separation combined with a
label-free approach and /E1 %rbitrap 7SA7S analysis. Subcellular fractionation was used prior
to proteomic analysis to isolate the chromatin fraction which is !nown to contain the "N$-bound
proteins, many of which are involved in the regulation of gene e#pression<a !ey mechanism in
the process of stem cell differentiation. )ractionation was also important for improving the
resolution, as the use of whole cell lysates to e#amine differentiating cells results in the dilution of
,'
the low abundance proteins. Dy isolating the chromatin fraction we were able to avoid the
mas!ing of these proteins by the higher abundance non-nuclear proteins. The use of labels,
although popular in previous studies, was avoided. $ label-free approach enabled us to greatly
increase the sensitivity 4and hence resolution5 of our e#periments, as well as ensuring unbiased
results. ?e also avoided the use of 2"-separation methods, preferring a +"-S"S-/$6. analysis
which offered a number of advantages over rival approaches, including greater reproducibility,
ease of use, and the ability to maintain the proteins in a low volume and high concentration state
which further improved the sensitivity. The +" gel-approach is also easily interfaced with the
mass spectrometry /E1 system we utilised.
Ising our approach we were able to quantitatively identify over 2,'(( proteins in the chromatin
proteome of differentiating NT2 cells. To our !nowledge, this is the deepest and highest-
resolution coverage of the NT2 chromatin proteome to date, as well as the first time that Nanog
has been identified in differentiating NT2 cells. The proteins identified included a large number of
low abundance proteins previously implicated in the regulation of pluripotency, such as %ct',
Nanog, and So#2.$ number of other transcription factors and chromatin remodelling comple#es
previously implicated with pluripotency were also identified in our dataset. These include the
comple#es Nu2", hS?3ASN), Draf-"$1 and Sin,-"$1, and the factors "ppa2 and "ppa',
Sall', Sall+, and 2if+, 2equiem, and $rid,b, to name but a few. 3n congruence with previous
studies, our results show that these proteins decrease in abundance as differentiation proceeds
=+,,+-,',,''>.
/uture applications and routes of en%uiry
$ subset of -' proteins was used to illustrate one of several potential future applications of our
optimised approach and the resulting dataset. ere, a protein interaction networ! was generated,
in silico to show the interaction of pluripotency transcription factors with chromatin remodelling
comple#es, and how the abundances of these proteins appear to be altered at specific stages of
,-
differentiation. The timing of this can be indicative of their role in the differentiation process
4)igure '.+5@ for instance if a protein is down-regulated in the early stages it is more li!ely to play
a role in the maintenance of FstemnessG. Similarly, if a protein is upregulated in the later stages it
could represent a protein specifically involved in the acquisition of a neuronal cell identity. The
sequence in which interacting proteins are downregulated can also provide insight into the
mechanisms through which their encoding genes are repressed@ in the networ! a number of
remodelling comple#esR subunits were downregulated prior to the pluripotency transcription
factors, potentially indicating that the alteration of their compositions plays a role in the switch
from a pluripotent, undifferentiated state to a lineage-specific differentiating state. 3n order to
conclusively tac!le these questions, further e#periments would have to be carried out, such as
e#amining the effect on pluripotency and differentiation if the subunits in question are !noc!ed
down or endogenously e#pressed through transfection with lentiviral vectors.
*igre !.1. E('othetical re'resentation of information 3hich can be derived throgh
anal(sis of the dataset.
,0
%ver +(( putative uncharacterised proteins were also discovered, providing the potential to
establish novel stem cell factors through further analyses. %ne possible route of enquiry would be
to select several of the top-ran!ing uncharacterised proteins and carry out 2N$i !noc!down
e#periments to disrupt the genes encoding them, measuring the effects of this on three
pluripotency-related phenotypesH al!aline phosphatase e#pression, cell proliferation rate, and
Nanog and %ct' e#pression. This could allow one to identify the functions of the proteins, and
determine if this function is relevant to pluripotency. )urthermore, the dataset could be e#amined
for proteins which are significantly upregulated during differentiation in order to attempt to
discover which proteins and signalling pathways are involved in the homogenous differentiation
of NT2 cells into neuronal cells. 6iven the current limited efficiency in directed differentiation of
stem cells for therapeutic purposes, such information could provide !ey insights into improving
techniques.
Conclusions
%verall, the study showed that a label-free quantitative proteomics approach could successfully
monitor changes in protein abundance over a stem cell differentiation cycle. 1hanges were found
in the abundance of protein comple#es involved in epigenetic regulation, such as Nu2", and
!nown stem cell transcription factors, such as Nanog. 6iven the comple#ity of cellular
differentiation and the large numbers of proteins involved, a proteomics approach may therefore
provide a useful overview of the cellular changes involved, and suggest new targets for analysis,
that ultimately may lead to better understanding of stem cells and perhaps feasible clinical
applications.
,8
Total 3ord cont: =+!7#
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